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Am J Physiol Heart Circ Physiol 282: H784-H792, 2002; doi:10.1152/ajpheart.00997.2000
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Vol. 282, Issue 2, H784-H792, February 2002

UDP acts as a growth factor for vascular smooth muscle cells by activation of P2Y6 receptors

Mingyan Hou1, T. Kendall Harden2, Cynthia M. Kuhn4, Bo Baldetorp6, Eduardo Lazarowski2,3, William Pendergast5, Sebastian Möller1, Lars Edvinsson1, and David Erlinge7

1 Division of Experimental Vascular Research, Department of Medicine, University Hospital, SE-221 85 Lund, Sweden; Departments of 2 Pharmacology and 3 Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599; 4 Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27710; 5 Inspire Pharmaceuticals Incorporated, Durham, North Carolina 27700; and Departments of 6 Oncology and 7 Cardiology, University Hospital, SE-221 85 Lund, Sweden


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mitogenic effects of the extracellular nucleotides ATP and UTP are mediated by P2Y1, P2Y2, and P2Y4 receptors. However, it has not been possible to examine the highly expressed UDP-sensitive P2Y6 receptor because of the lack of stable, selective agonists. In rat aorta smooth muscle cells (vascular smooth muscle cells; VSMC), UDP and UTP stimulated 3H-labeled thymidine incorporation with similar pEC50 values (5.96 and 5.69). Addition of hexokinase did not reduce the mitogenic effect of UDP. In cells transfected with P2Y receptors the stable pyrimidine agonist uridine 5'-O-(2-thiodiphosphate) (UDPbeta S) was specific for P2Y6 with no effect on P2Y1, P2Y2, or P2Y4 receptors. UDPbeta S stimulated [3H]thymidine and [3H]leucine incorporation and increased cell number in VSMC. Flow cytometry demonstrated that UDP stimulated cell cycle progression to both the S and G2 phases. The intracellular signal pathways were dependent on phospholipase C, possibly protein kinase C-delta , and a tyrosine kinase pathway but independent of Gi proteins, eicosanoids, and protein kinase A. The half-life of P2Y6 receptor mRNA was <1 h by competitive RT-PCR. The mitogen-activated protein kinase kinase inhibitor PD-098059 significantly suppressed, whereas ATP and interleukin-1beta upregulated, expression of P2Y6 receptor mRNA. The results demonstrate that UDP stimulates mitogenesis through activation of P2Y6 receptors and that the receptor is regulated by factors important in the development of vascular disease.

uridine 5'-diphosphate; gene expression


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE CARDIOVASCULAR EFFECTS of extracellular nucleotides have received increasing attention since the beneficial effects of the platelet inhibitory ADP receptor antagonist clopidogrel were demonstrated in atherosclerotic disease (2). Several other receptors for extracellular nucleotides (P2 receptors) have been cloned and found to be involved in other processes in cardiovascular regulation. Previous studies demonstrated that the extracellular nucleotides ATP and UTP act as growth factors for vascular smooth muscle cells (VSMC) by activation of P2Y1, P2Y2, and P2Y4 receptors (10, 14). However, it has not been possible to examine the UDP-activated P2Y6 receptor, which has the highest mRNA expression in the media of the rat aorta (11), because of the lack of selective agonists and antagonists.

UTP is present in all cells at ~10% of ATP levels and is formed through both salvage and de novo pathways. UTP can be released from both platelets and endothelial cells under physiologically relevant stimuli (1, 20, 30, 34). Released uridine nucleotides are catabolized by ectonucleotidases that are present on a variety of tissues and cells, suggesting that the concentration of extracellular UTP also reflects the concentration of extracellular UDP (9). So far, there is no assay for UDP quantification, so we do not know in what concentrations UDP is released. In vitro experiments with cultured cells demonstrated UTP release in a ratio of 1:5 compared with ATP (20), and in pathophysiological situations, extracellular ATP concentrations may temporarily exceed 100 µM in blood (8).

UDP has a contractile effect in blood vessels that is sometimes even as potent as UTP (30). UDP can induce the release of norepinephrine from sympathetic neurons and thromboxane A2 from cultured glia (19, 40). In a recent study in murine dendritic cells, it was observed that UDP could induce the release of cytokines (25).

So far, four subtypes of P2Y receptors have been cloned in the rat. Among these, P2Y2, P2Y4, and P2Y6 receptors can be activated by uridine nucleotides: P2Y6 receptors are activated by UDP, and P2Y2 and P2Y4 receptors are activated by UTP. The P2Y1 receptor, a purinoceptor, is only activated by ADP and ATP (30). Studies on cells transfected with P2Y6 receptors revealed that UDP, via activation of P2Y6 receptors, increases formation of inositol phosphates and Ca2+ concentration (7, 28). Molecular investigations showed that P2Y6 receptors are abundantly expressed in both contractile and synthetic phenotypes of VSMC and are slowly desensitized (11, 32).

The mitogenic effect of UDP was demonstrated previously (13), but no conclusion could be drawn as to whether this was mediated by the conversion of UDP to UTP during the experiment and thereby activation of P2Y2 or P2Y4 receptors. The availability of enzymatically stable pyrimidine analogs uridine 5'-O-(2-thiodiphosphate) (UDPbeta S) and uridine 5'-O-(3-thiotriphosphate) (UTPgamma s) has provided us an opportunity to determine whether UDP indeed acts as a growth factor for VSMC by activating P2Y6 receptors. Furthermore, we wanted to study whether P2Y6 receptors are regulated by factors that are important in the development of vascular disease.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996). A culture of aorta smooth muscle cells (VSMC) was prepared essentially as described previously (12). The aorta from adult Sprague-Dawley rats was removed under sterile conditions. With the use of a binocular microscope, the adventitia and outer media were stripped off. The vessels were opened by a longitudinal cut, and the intima was removed by scraping. The remaining media was cut into 1-mm pieces from which VSMC were cultured in DMEM containing 1,000 mg/l d-glucose, sodium pyruvate, L-glutamine, streptomycin (100 µg/ml), penicillin (100 U/ml), and 10% FCS at 37°C in humidified 5% CO2-95% air atmosphere for 5-16 passages. Cell viability was tested by exclusion of trypan blue (>95%). VSMC were identified by immunofluorescence staining of alpha -actin filaments with a monoclonal antibody (mouse IgG) against alpha -actin and a second anti-mouse antibody labeled with FITC (Boehringer-Mannheim). Examination of cell morphology excluded endothelial cell contamination.

Determination of DNA synthesis. DNA synthesis was measured by tritiated [3H] thymidine incorporation. VSMC cultures were suspended by trypsinization with trypsin-EDTA (1×), counted in a Burker hemocytometer chamber counter, and replated into 24-well plates at a density of 20,000 cells/well in medium (as described in Cell culture). After 24 or 48 h, the cells were starved in serum-deprived medium for another 48 h to decrease proliferation and induce quiescence. All the studied substances were added at the same time and were present for 19 h except for antagonists, which were added 1 h before, and pertussis toxin and phorbol 12-myristate 13-acetate (PMA), which were added 24 h before. [3H]thymidine (0.2 µCi/ml) was added during the last 4 h of stimulation. The medium was then aspirated, and the cells were washed twice with cold PBS and twice with ice-cold 10% trichloroacetic acid. The fixed cellular material was solubilized in 1 ml of 0.2 M NaOH for 2 h or overnight. The solution from each well (1 ml) was mixed with 4 ml of OptiPhase HiSafe 3 liquid scintillation cocktail. The amount of [3H]thymidine uptake was estimated by counting in a Wallac 1410 liquid scintillation counter.

Determination of cell number using colorimetric method. According to the instructions of the CellTiter 96 AQ One Solution Cell Proliferation Assay (Promega), the tetrazolium compound 3-(4,5-dimethythiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) is bioreduced by viable cells into colored formazan that is soluble in tissue culture medium. The conversion of MTS is accomplished by dehydrogenase enzymes found in living cells. The amount of formazan can then be determined by measuring absorbance at 490 nm in an ELISA plate reader. The quantity of formazan product, and thus the amount of 490-nm absorbance, is directly proportional to the number of living cells in culture.

Rat aorta VSMC were suspended by trypsinization, counted, replated into 96-well plates at a density of 2,000 cells/well in medium containing 10% FCS, and then starved for 48 h to induce quiescence. All the studied agonists were added at the same time and were present for 3 days. Twenty microliters of MTS reagent in one hundred microliters of DMEM were added after changing culture medium for another 4 h in the incubator before recording.

Protein synthesis. [3H]leucine incorporation was used for measuring protein synthesis. The method is similar to that described in Determination of DNA synthesis with the exception that 0.2 µCi/ml of [3H]leucine instead of radioactive thymidine was present in the medium for 96 h in the presence of the studied substances.

Flow cytometry. The staining of nuclear DNA content and flow cytometric analysis were performed as previously described (4, 5). Briefly, for staining of nuclei cells were washed in PBS, whereupon nuclei isolation medium containing propidium iodide was added. Samples were incubated in the dark for 5 min at room temperature. Samples were then kept at 4°C for at least 15 min before flow cytometric analysis was performed in an Ortho-Cytoron Absolute (Ortho Diagnostic Systems, Raritan, NJ) equipped with a 15-mW argon ion laser. The laser line at 488 nm was used for excitation of propidium iodide, and fluorescence beyond 620 nm was detected. Up to 20,000 nuclei/sample were analyzed. Processor signals were digitized and sorted into frequency distributions, DNA histograms, with a resolution of 256 units. Cell cycle phase distribution, i.e., the fractions of G0/G1, S, and G2 nuclei, of the analyzed cell population was determined by using Multi Cycle software (Phoenix Flow Systems, Tucson, AZ) on the DNA histograms. The DNA histograms were corrected for contribution of nucleic debris.

RNA extraction. The samples, which were stimulated with different studied substances in the indicated time, were used to extract RNA with TRIzol reagent (GIBCO BRL) following the supplier's instructions. The resulting RNA pellet was finally washed with 70% ice-cold ethanol, air-dried, and redissolved in 10 µl of diethylpyrocarbonate-treated water. The RNA concentration was determined spectrophotometrically. The quality of the RNA preparation was assessed by estimating the ratio of 18S to 28S rRNA on a denaturing gel.

Competitive RT-PCR. Competitive RT-PCR was carried out as described in detail previously (11). Specific primers for the rat P2Y6 receptor (7) were designed (forward, 5'-GTTATGGAGCGGGACAATGG-3'; reverse, 5'-AGGATGCTGCCGTGTAGGTT-3'), generating a PCR product of 347 bp. A synthetic RNA competitor for the P2Y6 receptor, bearing a deletion of 80 bp compared with the wild-type sequence, had been previously constructed, which generated a product of 267 bp (11). For competitive RT-PCR, 300 ng of total RNA was mixed with different amounts of competitor RNA in five subsequent 1:5 dilution steps. RT-PCR was carried out with a GeneAmp RNA PCR kit on a GeneAmp PCR system 2400 (Perkin-Elmer). First-strand cDNA synthesis was performed with the AmpliTaq RNA-PCR kit (Perkin-Elmer) in a 20-µl volume with the reverse primer for priming. Finally, amplification was performed with the following profile: 5 min at 95°C, followed by 45 cycles of 1 min at 95°C, 30 s at 59°C, 30 s at 72°C, and a final extension step of 7 min at 72°C. Because the studied P2Y6 receptor is intronless within its coding region, PCR without the RT step was always used to exclude genomic DNA contamination.

Densitometric analysis. Ten microliters of PCR product was run on a 2% agarose gel in 0.5% Tris-boric acid-EDTA, stained with ethidium bromide (1 µg/ml), photographed on a ultraviolet box using Polaroid 667 film, and digitized using a flatbed scanner. Further densitometric analysis was performed with NIH Image software (31). The wild type-to-competitor ratio was calculated by taking the differing lengths of the products into account and plotted on a log scale against competitor concentration. For each experiment, a standard curve was calculated using linear regression and the equivalence point (log ratio = 0) was determined. Copy numbers were calculated and expressed as molecules per microgram of total RNA.

Assay of inositol phosphate accumulation in P2Y receptor-expressing 1321N1 cells. The human P2Y1, P2Y2, P2Y4, and P2Y6 receptors were stably expressed individually in 1321N1 human astrocytoma cells with retroviral vectors as previously described (35). Cells were labeled overnight with [3H]inositol, and agonist-promoted [3H]inositol phosphate accumulation was quantitated after a 10-min incubation in the presence of 10 mM LiCl and the appropriate agonist for activation of each receptor.

Materials. DMEM and PBS were obtained from Sigma, FCS and penicillin-streptomycin from GIBCO, [3H]thymidine and [3H]leucine from Amersham, and platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin, wortmannin, K252a, GÖ-6976, RO-31-8220, RO-31-7549, NGIC, PMA, H-89, PD-098059, SB-203580, T-25, forskolin, and pertussis toxin from Calbiochem. Oligonucleotides were obtained from GIBCO BRL. Unless stated otherwise, all reagents were purchased from Sigma.

Statistical analysis. Results are presented as means ± SE. Differences between values were evaluated by unpaired Student's t-test [not significant (NS) = P > 0.05]. Multiple comparisons were evaluated with ANOVA. Data were analyzed with StatView software.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

UDP-stimulated DNA synthesis in VSMC. The following results are presented as the percentage above controls; thus 0% equals control and 100% represents a twofold increase over the control value.

UDP induced a concentration-dependent increase of [3H]thymidine incorporation (Emax = 68 ± 10% above control, pEC50 = 5.96). The maximum UDP effect was 90% of PDGF (1 ng/ml)-stimulated [3H]thymidine incorporation when added in parallel in the same 24-well plate (Table 1). The 19-h incubation time was chosen because the DNA synthesis in response to growth factors usually starts 15-20 h after stimulation. Experiments were also done with longer incubation times (2-4 days), but the results for [3H]thymidine incorporation were similar.

                              
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  		Table 1.   Effects of UDP in combination with other growth factors

UTP, UTPgamma S, and UDPbeta S stimulated [3H]thymidine incorporation in a pattern similar to that of UDP with no significant difference in Emax and pEC50 (UTP: Emax = 106 ± 16% above control, pEC50 = 5.69; UTPgamma S: Emax = 96 ± 18% above control, pEC50 = 5.59; UDPbeta S: Emax = 73 ± 17% above control, pEC50 = 5.42). There was no significant difference in the Emax or pEC50 values between UDP and UDPbeta S or between UTP and UTPgamma S.

To exclude a UTP-mediated effect secondary to phosphorylation of UDP, hexokinase, which degrades UTP to UDP, was used (28). There was no difference when the cells were incubated with UDP in the presence of hexokinase for 19 h compared with UDP alone (62 ± 26% vs. 54 ± 14% above control; Fig. 1B). At a concentration of 1 µM, UTPgamma S and UDPbeta S stimulated DNA synthesis in an additive way (67 ± 10% vs. 38 ± 8% or 36 ± 7% above control; P < 0.05; Fig. 1C).


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  		Fig. 1.   Accumulation of [3H]thymidine incorporation by pyrimidine nucleotides on rat aorta smooth muscle cells. A: quiescent cells were stimulated with different concentrations of agonists for 19 h. Increased DNA synthesis was measured by incubating [3H]thymidine for the last 4 h. UDPbeta S, uridine 5'-O-(2-thiodiphosphate); UTPgamma S, uridine 5'-O-(3-thiotriphosphate). B: growth-arrested cells were incubated with hexokinase (10 U/ml) for 1 h and then in the presence or absence of UDP (30 µM) for 19 h. C: additive effect of UTPgamma S (1 µM) and UDPbeta S (1 µM). Data are presented as % above control; n = 9-21 experiments run in triplicate. *P < 0.05, not significant (NS) = P > 0.05 (Student's t-test).

The amount of [3H]leucine incorporation was also increased when cells were treated with different agonists for 4 days at a concentration of 30 µM (UDP 42 ± 7%; UDPbeta S 25 ± 7%, UTPgamma S 25 ± 9%, UTP 46 ± 8% above control; Fig. 2).


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  		Fig. 2.   Stimulation of protein synthesis by pyrimidine nucleotides. After 48-h starvation, the growth-arrested cells were stimulated with UTP (30 µM), UTPgamma S (30 µM), UDP (30 µM), and UDPbeta S (30 µM) for another 96 h; n = 16 experiments run in triplicate or quintuplicate. Data are presented as % above control. NS = P > 0.05 (Student's t-test).

Cell cycle. After 72 h of stimulation with UDP, UDPbeta S, UTP, and UTPgamma S, the cell number was increased, with the maximal effect at a concentration of 30 µM (Fig. 3A). To further support that UDP can stimulate the progression through the whole cell cycle, flow cytometry was used. There was a significant increase of cell percentage in both the S phase and the G2 phase after 19 h of stimulation with 30 µM UDP (Fig. 3B).


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  		Fig. 3.   Increase of cell number and completion of cell cycle by pyrimidine nucleotides. A: cells were treated with different agonists at a concentration of 30 µM for 3 days. The cell number was quantified with a colorimetric method. Data are presented as % above control; n = 15 experiments run in quintuplicate. B: growth-arrested cells were stimulated with 30 µM UDP for 19 h, and then the amount of cells at different stages of the cell cycle was measured as described in MATERIALS AND METHODS. Data are presented as % of total cells; n = 6. ***P < 0.001 (Student's t-test).

Effects of UDP in combination with other growth factors. Several growth factors such as PDGF, basic fibroblast growth factor (bFGF), and EGF can be released under pathophysiological conditions. To determine whether they can modulate the effect of UDP on [3H]thymidine incorporation, we stimulated cells with UDP combined with growth factors. As shown in Table 1, minor synergistic effects were observed in the combination of UDP with PDGF but only additive effects in combination with bFGF and EGF.

Inhibition of phospholipase C. The phospholipase C (PLC) inhibitor U-73122 (1 µM) significantly attenuated the accumulation of [3H]thymidine incorporation by UDP (233 ± 20% vs. 160 ± 22%).

Inhibition of protein kinases. K252a, which inhibits both cyclic nucleotide-dependent protein kinases and protein kinase C (PKC), totally blocked the DNA synthesis induced by UDP from 187% above control to 79% below control. However, this effect seems to be nonspecific because the basal levels of DNA synthesis were also significantly inhibited (84 ± 3% below control). When the concentration of K252a used was one log unit lower, there was no inhibition by K252a of the DNA synthesis induced by UDP (data not shown). Neither GÖ-6976, an inhibitor of Ca2+-dependent PKC-alpha and -beta 1 isoforms, nor RO-31-8220, an inhibitor of PKC-alpha , -beta , -gamma , and -epsilon isoforms, blocked the effect of UDP on DNA synthesis (GÖ-6976: 81 ± 15% vs. 72 ± 13%; RO-31-8220: 135 ± 23 vs. 102 ± 15%) (26, 39). When GÖ-6976 and RO-31-8220 were combined, there was still no inhibition (data not shown). When the concentrations of the above-described blockers were increased one or two log units, there was either no inhibition or nonspecific inhibition (data not shown). However, the prolonged incubation of PMA (1 µM), which downregulates PKC-delta isoforms (37), totally attenuated the accumulation of [3H]thymidine incorporation by UDP without any effects on the basal levels (155 ± 18% above control vs. 7 ± 15% below control; Table 2). The selective protein kinase A inhibitor H-89 had no effect on [3H]thymidine accumulation by UDP (128 ± 20% vs. 119 ± 17% above control; Table 2).

                              
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  		Table 2.   Effects of protein kinase inhibitors on accumulation of [3H]thymidine incorporation induced by UDP

Inhibitory effect of cAMP on DNA synthesis by UDP. Intracellular cAMP inhibited DNA synthesis in our study, because the adenylyl cyclase activator forskolin (3 µM) significantly inhibited both basal levels (59 ± 4% below control) and UDP-induced accumulation (148 ± 15% above control vs. 24 ± 6% below control) of [3H]thymidine incorporation. Pretreatment with pertussis toxin (50 ng/ml) for 24 h did not inhibit UDP-stimulated mitogenesis (203 ± 32% vs. 168 ± 25% above control). Indomethacin (1 µM), a cyclooxygenase inhibitor, did not block the effect of UDP on the accumulation of [3H]thymidine incorporation (294 ± 29% vs. 228 ± 19% above control).

Inhibitors of mitogen-activated kinases. The mitogen-activated protein kinase (MAPK) kinase (MAPKK) inhibitor PD-098059, which inhibited basal levels of [3H]thymidine incorporation by 35% below control, significantly blocked the accumulation of [3H]thymidine incorporation by UDP (186 ± 26% vs. 93 ± 22% above control). In a similar nonspecific manner, another MAPK subgroup P38 blocker, SB-203580 (1 µM), nonspecifically inhibited DNA synthesis induced by UDP (UDP vs. UDP + SB-203580: 210 ± 44% above control vs. 35 ± 8% below control; SB-20358 alone: 57 ± 17% below control). The tyrosine kinase inhibitor tyrphostin 25 had no effect on the basal levels of [3H]thymidine incorporation, but it significantly abolished the accumulation of [3H]thymidine incorporation induced by UDP, indicating specific inhibition (198 ± 46% above control vs. 5 ± 9% below control).

Regulation of P2Y6 receptor mRNA expression. Actinomycin D was introduced to the growth-arrested cells for the indicated time to study the half-life of P2Y6 receptor mRNA. The level of P2Y6 receptor mRNA was rapidly degraded within 1 h, suggesting that the half-life of P2Y6 receptor mRNA is <1 h (~46 min). The degradation of P2Y6 receptor mRNA was further increased at 6 and 24 h (Fig. 4A).


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  		Fig. 4.   Regulation of mRNA by different agents. A: time-dependent degradation of P2Y6 receptor mRNA. Quiescent cells were stimulated with 5 µg/ml actinomycin D for the times indicated. B: downregulation of P2Y6 receptor mRNA by the extracellular signal-regulated kinase-1/2 inhibitor PD-098059 (30 µM), which was introduced to cells for 24 h. C: upregulation of P2Y6 receptor mRNA by 30 µM ATP and 10 ng/ml interleukin-1beta , which were used to stimulate cells for 24 h. The levels of P2Y6 receptor mRNA were measured by competitor RT-PCR and are presented as % of control; n = 3-6. **P < 0.01; ***P < 0.001 (Student's t-test).

The essential role of MAPKK has been established for the expression of P2Y2 receptor mRNA (17). To examine whether MAPKK plays the same role in the expression of P2Y6 receptor mRNA, the selective MAPKK inhibitor PD-098059 was used. PD-098059 significantly inhibited the basal level of P2Y6 receptor mRNA expression (20 ± 8% above control; Fig. 4B).

Under pathophysiological conditions, a number of factors such as interleukin (IL)-1beta and ATP can be released that have been found to upregulate P2Y2 receptors (17). Therefore, in this study we examined their effects on the expression of P2Y6 receptor mRNA. The amount of P2Y6 receptor mRNA was significantly increased up to 240% of control on the cells treated with ATP for 24 h. IL-1beta also significantly increased the levels of P2Y6 receptor mRNA (522 ± 108% of control; Fig. 4C).

Effect of UDPbeta S on inositol phosphate accumulation in P2Y receptor-expressing 1321N1 cells. Concentration effect curves were generated for UDPbeta S and UDP for stimulation of [3H]inositol phosphate accumulation in 1321N1 cells stably expressing the human P2Y6 receptor (Fig. 5A). These nucleotides stimulated [3H]inositol phosphate accumulation to similar maximal levels and with similar EC50 values [EC50 = 28 ± 13 nM (mean ± SE of n = 5 experiments) for UDPbeta S; EC50 = 47 ± 15 nM (mean ± SE of n = 4 experiments) for UDP]. Although UDPbeta S was at least as active as the cognate agonist UDP for the P2Y6 receptor, it had no effect on [3H]inositol phosphate accumulation in 1321N1 cells stably expressing the ADP-selective P2Y1 receptor, the UTP and ATP-selective P2Y2 receptor, or the UTP-selective P2Y4 receptor (Fig. 5, B and C). Thus UDPbeta S is a potent selective agonist for the study of the P2Y6 receptor.


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  		Fig. 5.   Concentration effect curves for UDPbeta S and UDP for stimulation of [3H]inositol phosphate accumulation in 1321N1 cells stably expressing the human P2Y6 receptor (A). Although UDPbeta S was as least as active as the cognate agonist UDP for the P2Y6 receptor, it had no effect on [3H]inositol phosphate accumulation in 1321N1 cells stably expressing the ADP-selective P2Y1 receptor (B), the UTP- and ATP-selective P2Y2 receptor (C), or the UTP-selective P2Y4 receptor (D).

Susceptibility to degradation. Primary cultures of human nasal epithelial cells were incubated with ~300 000 cpm/ml of either 30 µM UDPbeta S or 30 µM UDP in the medium (0.25 ml). At the times indicated, the medium was harvested and 3H-labeled nucleotides were measured with HPLC. As seen in Fig. 6, UDP was rapidly degraded, whereas UDPbeta S was resistant to degradation during the 2-h incubation period.


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  		Fig. 6.   Susceptibility of UDP and UDPbeta S to the hydrolytic action of 1321N1 astrocytoma cell nucleotidases. At the times indicated, the medium was collected and nucleotides were quantified by HPLC. Data represent n = 2-4 experiments for each time point.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previous studies demonstrated that the mitogenic effects of the extracellular nucleotides ATP and UTP are mediated by P2Y1, P2Y2, and P2Y4 receptors in VSMC (10, 14). Although UDP has been reported to stimulate VSMC proliferation, it is not clear whether this effect is mediated by P2Y6 receptors because UDP can be phosphorylated by phosphatase enzymes to UTP (13, 21, 28). Furthermore, with quantitative competitive RT-PCR, it was found that all the P2Y receptors were detected in VSMC in the vessel wall and that P2Y6 receptors were most abundantly expressed (11, 13). The P2Y6 receptor is of special interest because it has a different COOH-terminal domain, making it resistant to desensitization by long-term stimulation. Therefore, we wanted to establish whether P2Y6 receptors mediate mitogenic effects of UDP in VSMC.

Our results showing that UDP and UTP stimulated [3H]thymidine incorporation and cell number with equal potency cannot be explained by only one of the P2Y receptors cloned so far. Given our previous knowledge that UTP stimulates mitogenesis in these cells through P2Y2 and P2Y4 receptors, the results indicate that UDP mediates its effects through activation of P2Y6 receptors. However, UDP could also mediate its effect via P2Y2 and P2Y4 receptors after its conversion to UTP. To address this problem, we used two approaches.

First, we used hexokinase, which degrades UTP to UDP, thereby removing UTP and preventing the generation of UTP caused by phosphorylation of UDP (28). Cells transfected with the P2Y6 receptor showed equal potency for UDP alone and UDP combined with hexokinase, whereas cells transfected with P2Y2 and P2Y4 receptors showed a decreased response to UDP when it was combined with hexokinase (21, 28). We found that hexokinase did not affect the stimulation of DNA synthesis induced by UDP, suggesting the involvement of P2Y6 receptors in the proliferation of VSMC.

Second, we used the stable pyrimidines UTPgamma S and UDPbeta S, in which a thio substitution at the terminal phosphates has been shown to provide stability to ectonucleotidase action (22). In cells transfected with P2Y2, P2Y4, or P2Y6 receptors, UTPgamma S has been shown to selectively activate P2Y2 and P2Y4 receptors with no effects on P2Y6 receptors (22, 28). UDPbeta S, on the other hand, is a specific agonist for P2Y6 receptors with no effect on P2Y1, P2Y2, or P2Y4 receptors, as seen in Fig. 5. Furthermore, it was important to show that UDPbeta S was resistant to degradation by various phosphatases and nucleotides. The most adequate analysis is by comparative studies in the presence of tissue. As seen in Fig. 6, although UDP was rapidly metabolized in the presence of airway tissue, UDPbeta S was unchanged after prolonged incubation. This demonstrates that UDPbeta S is a stable and selective agonist for P2Y6 receptors.

The mitogenic effects of UDP and UTP were equally potent as the stable pyrimidines UDPbeta S and UTPgamma S, suggesting that in our cultured VSMC there is not any major interconversion between UTP and UDP, i.e., neither any important degradation nor any conversion of UDP into UTP. This is in contrast to intact arteries, in which the abundant presence of ectonucleotidases makes the stable pyrimidines far more potent than UTP and UDP (24), but it is similar to the responses in stably transfected 1321N1 astrocytoma cells, confirming that intact tissue has markedly higher ectonucleotidase activity than cell cultures.

The similar potencies of UDPbeta S and UTPgamma S indicate involvement of more than one P2Y receptor subtype. The mitogenic effects of UTPgamma S are mediated by P2Y2 and P2Y4 receptors as shown previously for UTP (10, 12, 14). However, there is no known receptor other than the P2Y6 receptor that is activated by UDPbeta S. Thus the results strongly suggest that UDPbeta S and UDP act as growth factors for VSMC by activating the P2Y6 receptor.

Growth factors can be divided into competence growth factors that stimulate cells to go from G0 to G1 phase and progression growth factors that can stimulate cells to go from G1 to S and M phases. Most polypeptide growth factors, which play an essential role on the formation of atherosclerosis and neointima, are dependent on progression growth factors to exert their proliferative effects (18). The extracellular nucleotides ATP and UTP have been demonstrated to act as progression growth factors via the P2Y2 receptor in VSMC (27). The significant increase of total cell number induced by extracellular UDP indicates that UDP may stimulate the progression through the whole cell cycle per se. This was confirmed by using flow cytometry in which UDP stimulated the progression to both the S and the G2 phase. This suggests that UDP is a potent growth factor that acts both as a competence growth factor and as a progression growth factor via P2Y6 receptors with the ability to take the serum-deprived G0-arrested cells through the whole cell cycle. However, synergism with other growth factors produced in an autocrine manner by the VSMC cannot be excluded.

Intracellular mechanisms. It is well known that Gq protein-coupled receptors activate PLC, stimulating the synthesis of diacylglycerol, inositol phosphate formation, and intracellular Ca2+ release. Consistent with this, U-73122 specifically inhibited DNA synthesis induced by UDP, suggesting the involvement of PLC in the accumulation of [3H]thymidine incorporation by UDP via P2Y6 receptors. This is supported by our finding that UDP increased intracellular Ca2+ (data not shown) and the stimulation of inositol phosphate formation by UDP and UDPbeta S in cells transfected with the P2Y6 receptor.

PKC consists of at least 11 closely related isoforms (15). With the use of Western blots and RT-PCR, PKC-alpha , -delta , -zeta , and -epsilon isoforms were detected in smooth muscle cells, but their expression varied in various states of differentiation (3). Because the general PKC downregulator PMA specifically inhibited the mitogenic response, whereas the inhibitors of PKC-alpha , -zeta , and -epsilon were without effect, it is possible that UDP acts via PKC-delta to transmit signal to the nucleus. Indeed, one study has demonstrated that in response to P2Y2 receptors PKC-delta can be phosphorylated on tyrosine residues (37). This phosphorylation pathway fits well with the presently studied P2Y6 receptors because tyrphostin 25 and prolonged incubation of PMA specifically inhibited the mitogenic effect of UDP (Table 2). However, we cannot make a firm conclusion on a role for PKC-delta in the PKC-dependent signaling pathway induced by UDP because prolongation of PMA also inhibits activation of MAPK (37).

In 1993, Shigeri and Fujimoto (36) observed that G protein-coupled receptor induced DNA synthesis that involved regulation of adenylate cyclase activation. Studies in other cell types indicate that P2 receptors can inhibit accumulation of cAMP via Gi proteins (29, 38). P2Y6 receptors are not known to couple to cAMP (6). In agreement with this, we found that pertussis toxin did not block the mitogenic effect of UDP. Furthermore, cAMP-dependent protein kinase A is not involved in the DNA synthesis induced by UDP because H-89 had no effect on the accumulation of [3H]thymidine incorporation by UDP. Indomethacin did not inhibit DNA synthesis induced by UDP, indicating that prostaglandins are not involved in the UDP-induced mitogenic effect.

It is established that G protein-coupled receptors regulate MAPK cascades, leading to activation of the extracellular signal-regulated kinases (ERKs), Jun amino-terminal kinase/stress-activated protein kinase, and P38 MAPK (23). The concentrations of PD-098059 and SB-20358 used here were based on previous studies in which they were shown to selectively inhibit ERK-1/2 and P38 MAPK. In our VSMC these concentrations inhibited basal DNA synthesis, indicating a constant activation of these pathways even in serum-starved VSMC. Lower concentrations had no effect either on basal or UDP-stimulated [3H]thymidine incorporation.

The basal inhibitory effects of these selective inhibitors make it difficult to discriminate between the intracellular pathways beyond the second messengers. However, the tyrosine kinase inhibitor tyrphostin 25 specifically inhibited effects of UDP, indicating an essential role of a tyrosine kinase pathway in the signal pathway of mitogenic effects of UDP.

Regulation of P2Y6 receptor mRNA expression. We have extensively studied (11, 16, 17) how the mitogenic UTP-stimulated P2Y2 receptor is regulated in VSMC. The most important regulators were MAPKK-dependent growth factors and cytokines. To examine this for the P2Y6 receptor, mRNA levels were measured with a quantitative competitive RT-PCR recently developed in our laboratory (11).

Incubation with the RNA polymerase II inhibitor actinomycin D demonstrated that the P2Y6 receptor mRNA was rapidly degraded with a half-life of <1 h in a similar pattern as the half-life for P2Y2 receptor in VSMCs (17). This rapid turnover rate could facilitate regulation of P2Y6 receptor expression in pathophysiological situations. We previously showed (17) that continuous activation of MAPKK is necessary for the expression of P2Y2 receptor mRNA. Here we observed that MAPKK plays the same role on the expression of P2Y6 receptor mRNA because the basal levels of P2Y6 receptor mRNA were significantly inhibited by PD-098059. ATP, which can be released from various cardiovascular sources under pathophysiological and physiological conditions, upregulated P2Y6 receptor mRNA. It is possible that this can synergistically increase the effects of extracellular nucleotides by upregulating P2Y6 receptors, leading to increased vessel tone and VSMC proliferation. Atherosclerosis is considered as an inflammatory disease (33), and cytokines are potent stimulators of P2Y2 receptor expression (16). The inflammatory mediator IL-1beta markedly increased the P2Y6 receptor mRNA expression. Thus factors known to be involved in cardiovascular pathophysiology may upregulate P2Y6 receptors that may contribute to formation of atherosclerosis and neointima.

In summary, extracellular UDP stimulates DNA synthesis via P2Y6 receptors, which are pertussis toxin-insensitive Gq-coupled receptors. The intracellular signal pathways are dependent on PLC, possibly PKC-delta , and a tyrosine kinase pathway but independent of Gi proteins, eicosanoids, and protein kinase A. P2Y6 receptor mRNA expression is MAPKK dependent, has a rapid turnover rate, and is upregulated by ATP and IL-1beta . The findings suggest that P2Y6 receptors could be of importance in the regulation of vascular smooth muscle growth and differentiation.


    ACKNOWLEDGEMENTS

This study was supported by the Swedish Heart and Lung Foundation, the Swedish Hypertension Society, the Tore Nilsson Foundation, the Thelma Zoegas Foundation, the Jeansson Foundation, and Swedish Medical Research Council Projects 13130 (to D. Erlinge) and 5958 (to L. Edvinsson).


    FOOTNOTES

Address for reprint requests and other correspondence: D. Erlinge, Dept. of Cardiology, Lund Univ. Hospital, SE-221 85 Lund, Sweden (E-mail: david.erlinge{at}kard.lu.se).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpheart.00997.2000

Received 25 October 2000; accepted in final form 16 October 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 282(2):H784-H792
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