Introduction

Summary Introduction Materials & Methods Results Discussion References

Extracellular ATP elicits functional responses in many cell types by activating P₂-purinergic nucleotide receptors (1); these include both G protein-coupled nucleotide receptors (collectively termed the P_2Y class) and ionotropic ATP-gated channel receptors (termed the P_2X class). Each of these major classes comprises a number of pharmacologically and genetically distinct receptor subtypes; cDNAs or genes encoding at least six different P_2Y class receptors (2) and seven different P_2X class receptors (3) have been recently cloned. Despite the growing number of distinct ATP receptor subtypes with redundant signaling properties, few studies have addressed issues regarding the factors that regulate the cell-specific expression of these receptor subtypes.

The P_2Y receptor family includes the P_2UR (also termed P2Y₂ by recommended IUPHAR nomenclature), a subtype for which ATP and UTP are equipotent agonists. In the presence of micromolar ATP or UTP, P_2UR activate PI-PLC effector enzymes, rapid mobilization of InsP₃-sensitive Ca²⁺ stores, and enhanced Ca²⁺ influx (2, 4, 5). Depending on the cell type, P_2UR can activate PI-PLC enzymes via the mediation of either the G_i or G_q family of G proteins (1, 2). Cloned DNAs encoding P_2UR have been isolated from several species and sources (2), including murine neuroblastoma cells (6), human epithelial cells (7), and rat genomic DNA (8). All of these cDNAs are highly homologous. Northern blot analysis and functional studies have indicated that P_2UR are expressed in a wide range of tissues. However, the organization and promoter sequences of P_2UR genes have not been reported, and little is known concerning the regulation of P_2UR expression.

We and others have previously reported that P_2UR are expressed in most myeloid leukocytes, including neutrophils, monocytes, macrophages, and the myeloid progenitor cells in marrow (9-12). Myeloid leukocytes provide a useful model for studying developmental regulation of ATP receptor expression because these cells are continuously replenished throughout adult life during two major stages of development and differentiation. Myeloid progenitor cells differentiate in the bone marrow over the course of several days to yield the circulating blood granulocytes and monocytes. However, blood monocytes and tissue macrophages (which are derived from monocytes) exist as only partially differentiated, quiescent cells until activated by immune or inflammatory stimuli. Thus, inflammatory activation of monocytes/macrophages represents the second stage of myeloid differentiation, which is characterized by major changes in gene expression and the induction, or repression, of signaling proteins involved in regulation of the inflammatory response (13). This raises the possibility that ATP receptors might also act as inducible or repressible signaling proteins whose expression can be regulated at the transcriptional/translational levels during myeloid differentiation and inflammatory activation.

In this study, we investigated the regulation of P_2UR expression in HL-60 cells, a human promyelocyte line derived from a patient with M3 acute myelogenous leukemia. These progenitor cells have been extensively used as an in vitro model for myeloid differentiation (14). When cultured in the presence of dibutyryl cAMP or DMSO, these cells assume a granulocyte/neutrophil phenotype. In contrast, treatment with phorbol esters induces HL-60 cells to acquire many phenotypic features characteristic of inflammatory monocytes and macrophages. These studies indicate that the expression of both P_2UR mRNA and functional P_2UR is rapidly and significantly modulated during these programs of in vitro myeloid differentiation.

	Materials and Methods

Summary Introduction Materials & Methods Results Discussion References

Cell culture. The HL-60 and THP-1 human leukocyte cell lines (American Type Culture Collection, Rockville, MD) were routinely maintained in Iscove's modified minimal essential medium medium (GIBCO, Grand Island, NY) with 10% iron-supplemented calf serum (Hyclone Laboratories, Logan, UT) in a humidified atmosphere of 92.5% air/7.5% CO_2, at densities between 3 × 10⁵ and 1 × 10⁶/ml. For granulocytic differentiation, HL-60 cells were transferred to serum-free Iscove's medium supplemented with transferrin, insulin, selenium, 2 mM glutamine, 1 mg/ml BSA, 100 units/ml penicillin, and 100 µg/ml streptomycin for 24 hr before induction with 500 µM Bt₂cAMP. Where indicated, HL-60 cells were also treated with 100 nM PMA or 5 µg/ml actinomycin D (Boehringer-Mannheim Biochemicals, Indianapolis, IN) added directly to serum-containing growth medium. In certain experiments, THP-1 promonocytes were differentiated into inflammatory macrophages by treatment with 1000 units/ml recombinant human IFN- (Genentech, South San Francisco, CA) and/or 1 µg/ml bacterial LPS (Escherichia coli 0111:B4; List Biomedicals, Campbell, CA) for 24-48 hr. A431 cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 2 mM glutamine at 5% CO₂. Human keratinocyte primary cultures, prepared from foreskin samples, were generously provided by Drs. Richard Eckert and Jean Welter (Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH). These cells were passaged three times, allowed to reach 70% confluence, and then treated with PMA or actinomycin D as indicated.

Measurement of cytosolic [Ca²⁺]. Adherent HL-60 cells from PMA-treated cultures were removed by washing in Ca²⁺ and Mg²⁺-free Hanks' balanced salt solution, followed by incubation in this solution with 300 µM EDTA for 10 min at 37°. Nonadherent, suspended cells were removed from growth medium through centrifugation. All cell types were then washed and resuspended at 1 × 10⁶/ml in a basal salt solution containing 125 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1.5 mM CaCl₂, 25 mM Na-HEPES, pH 7.5, 5 mM glucose, and 1 mg/ml BSA. Cells were incubated with 500 nM Fura-2-acetoxymethyl ester ester (Molecular Probes, Eugene, OR) for 40 min at 37°, centrifuged, resuspended in fresh medium at 3.3 × 10⁶/ml, and then incubated for an additional 10 min at 37°. Cells were stored on ice for 4 hr during measurements. Fura-2-loaded cells were assayed at 1.1 × 10⁶/ml in 1.5 ml in a stirred quartz cuvette at 37°. Fura-2 fluorescence was measured using 339 nm excitation and 500 nm emission. Where indicated, cells were incubated with 100 nM PMA and/or 300 nM staurosporine for 15 min at 37° before assay. Cells were lysed with 20 µg/ml digitonin for calibration as described previously (4).

Isotopic labeling of inositol phospholipids. HL-60 cells were removed from growth medium and resuspended at 1 × 10⁶/ml in serum-free, inositol-free Iscove's medium supplemented with insulin, transferrin, selenium, 2 mg/ml BSA, 4 mM glutamine, 50 units/ml penicillin, 50 µg/ml streptomycin, and 2 µCi/ml L-myo-[2-³H]inositol (American Radiolabeled Chemicals, St. Louis, MO) and incubated for 72 hr before experiments.

Measurement of inositol phosphate accumulation in intact HL-60 cells. Labeled cells were washed twice with basal salt solution. The cells were resuspended at 5 × 10⁶/ml in this buffer, and 0.2-ml aliquots were preincubated for 5 min at 37° before the addition of nucleotide agonists. After 15 sec, the reactions were terminated, and the samples were processed for analysis of InsP₂ and InsP₃ content as described previously (5).

Measurement of [³H]inositol phosphate production by isolated HL-60 cell membranes. Membranes from HL-60 cells were prepared as described previously (5). Briefly, cells were washed twice with an ice-cold buffer solution, resuspended in cold lysis buffer, and lysed by N₂ cavitation. EGTA-supplemented lysates were subjected to centrifugation, and final pellets were resuspended in cold EGTA-containing lysis buffer to yield stock membrane suspensions. Analysis of inositol polyphosphate production was performed exactly as reported previously (5). Briefly, aliquots of stock membrane suspensions were added to 37° assay buffer with the indicated concentrations of free Ca²⁺ and nucleotides. The reaction mixture was incubated at 37° for 5 min before organic extraction and analysis of [³H]InsP₂ and [³H]InsP₃ accumulation in the aqueous phase.

Northern blot analysis. Total RNA was extracted from cultured cells according to the method of Chomczynski and Sacchi (15). Poly(A)⁺ RNA was selected on oligo(dT) cellulose columns, precipitated, dissolved, and quantified by UV spectrophotometry. Then, 3.0 µg of poly(A)⁺ RNA/lane was electrophoresed on formaldehyde agarose gels. Gels were transferred to Nytran membrane by TurboBlotter rapid downward transfer (Schleicher & Schuell, Keene, NH) or by electroblotting in Tris/acetate/EDTA buffer (40 mM Tris/acetate, pH 8, 1 mM EDTA). The human P_2UR cDNA probe (SacI/BglII 814-1369 fragment) or cDNA probes corresponding to the FPR, IL-1, myeloperoxidase, and GAPDH gene products were random primer-labeled (Boehringer-Mannheim) with [-³²P]dCTP (Amersham). Blots were hybridized with probes by incubation in Quik-Hyb solution (Stratagene) for 1 hr at 65°. The blots were then washed and processed by standard methods. Hybridization of ³²P probes to specific bands was quantified using a Molecular Dynamics PhosphorImager (Sunnyvale, CA). Blots were stripped by boiling in 0.1× standard saline citrate (1× = 150 mM NaCl, 15 mM sodium citrate, pH 7.4) /0.1% sodium dodecyl sulfate before subsequent probing with other labeled cDNAs. The FPR cDNA was a generous gift from Dr. Daniel Perez (Department of Medicine, University of California, San Francisco, CA).

Semiquantitative RT-PCR. Total RNA was isolated by the above methods or by using a Qiagen (Studio City, CA) RNeasy total RNA kit. RNA (1.0 µg) was reverse-transcribed to cDNA in a 20-µl reaction volume containing 0.5 µg of oligo(dT) primer, 8 mM concentration of dNTPs, 40 units of RNasin (Boehringer-Mannheim), 10 mM MgCl₂, and 25 units of avian myeloblastosis virus RT (Boehringer-Mannheim) dissolved in a RT buffer (Promega, Madison, WI). The reactions were incubated for 1 hr at 42°, stopped by boiling for 2 min, and then diluted to 100 µl with sterile RNase-free water. Parallel aliquots of RNA samples (from control cells in each experiment) were subjected to mock RT reactions. These samples were incubated and prepared as described above, but no avian myeloblastosis virus RT was included. Diluted aliquots from the bona fide or mock RT reactions were then used as templates for PCR with primers specific to the human P_2UR (sense, 5-CTC TAC TTT GTC ACC ACC AGC GCG-3; antisense, 5-TTC TGC TCC TAC AGC CGA ATG TCC-3), generating the predicted 632-bp product. Commercial primers to GAPDH, IL-1, TNF-, and the human FPR (Stratagene) were also used to generate 600-, 332-, 355-, and 410-bp products, respectively. P_2UR and FPR reactions included 1.0 µM concentration of each primer, 0.8 mM concentration of dNTPs, 60 mM Tris·HCl, pH 8.5, 15 mM (NH₄)₂SO₄, 3.5 mM MgCl₂, and 1.25 units of Taq polymerase (Boehringer-Mannheim or United States Biochemical, Cleveland, OH) in a 50-µl reaction volume that was preincubated with 275 ng of TaqStart antibody 5-30 min at room temperature (Clonetech, Palo Alto, CA). TNF- reactions contained the same components but with 2.5 mM MgCl₂. GAPDH and IL-1 reactions included 1.0 µM concentration of each primer, 0.8 mM concentration of dNTPs, 10 mM Tris·HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 0.001% gelatin, and Taq polymerase pretreated as above. The PCR cycling protocols for each primer set were as follows: P_2UR and IL-1, 1 min at 94°, 2 min at 55°, and 4 min at 72°; GAPDH, 1 min at 94°, 2 min at 60°, and 2 min at 72°; FPR, 45 sec at 94°, 45 sec at 60°, and 1.5 min at 72°; and TNF-, 45 sec at 94°, 45 sec at 54°, and 1.5 min at 72°. Each protocol was carried out for 35 cycles and included an initial 5-min denaturation at 94° and a final 7-min extension at 72°. [-³²P]dCTP (0.1-0.4 µCi) was included in some PCRs to permit quantification of ³²P-labeled PCR products. These samples were quantified in duplicate where indicated. Ten microliters of each PCR product was electrophoresed on 1% agarose gels containing ethidium bromide. Gels were photographed, and each PCR product band was excised with the use of a razor blade and transferred to glass scintillation vials. Agarose slices of equal size were cut from unloaded lanes to measure background cpm. Slices were melted in 1.6 ml of water and counted for 1 min in 15 ml of scintillation fluid. Standard curves were generated using serial dilutions of the RT reactions as templates for PCR with each primer set. These curves were used to determine the linear range of the assay for each primer set and showed the assay to be sensitive to 2-fold changes in template level. Products from the original 20-µl RT reaction volumes were appropriately diluted into the final PCR volumes to ensure nonsaturation of the PCR amplification reactions: 1:50 or 1:100 dilutions were used for P_2UR analysis, 1:500 dilutions for the GAPDH analysis, and 1:50 dilutions for the FPR, TNF-, and IL-1 analyses. Primary data are presented as the amount of cpm of ³²P incorporated in each PCR product after subtraction of background radioactivity. Where indicated, data for P_2UR mRNA levels have been normalized relative to the amount of amplified GAPDH product. The absolute amount of radioactivity associated with given PCR products varies among experiments due to the use of [³²P]dCTP preparations with different specific activities.

	Results

Summary Introduction Materials & Methods Results Discussion References

Differential attenuation of P_2UR functional activity during granulocytic versus monocytic differentiation of HL-60 cells. In previous studies (4), we reported that functional activities of P_2UR were similar in both undifferentiated HL-60 promyelocytes and in HL-60 cells differentiated into granulocytes by treatment with Bt₂cAMP. In contrast, a very significant attenuation of P_2UR functional activity was observed in HL-60 cells treated with PMA, an agent that induces differentiation along the monocyte/macrophage pathway. Fig. 1, A-C, shows a comparison of the potency of UTP as a Ca²⁺-mobilizing agonist in undifferentiated HL-60 promyelocytes versus HL-60 cells treated with PMA for 2 days. Equivalent and maximal Ca²⁺ mobilization was observed when undifferentiated cells were stimulated with UTP concentrations of >3 µM (Fig. 1A). In cells treated with PMA for 48 hr, 3 µM UTP elicited no Ca²⁺ mobilization, and the response to 300 µM UTP, a normally supramaximal concentration, was greatly reduced (Fig. 1B). Concentration-response plots (Fig. 1C) indicated that both the potency and efficacy of UTP were significantly attenuated in PMA-differentiated HL-60 cells. Fig. 1C indicates that a 1-day treatment with PMA was also sufficient to induce a 2-log unit decrease in UTP potency. Because Ca²⁺ mobilization is secondary to the activation of PI-PLC-, the primary effector enzyme for these G protein-coupled P_2UR, we also compared UTP-induced accumulation of inositol polyphosphates in undifferentiated and PMA-differentiated HL-60 cells (Fig. 1D). Consistent with the Ca²⁺ mobilization data, both the potency and efficacy of UTP as an activator of PI-PLC were greatly reduced in PMA-differentiated cells.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   UTP-induced Ca2+ mobilization and inositol polyphosphate generation in control and PMA-differentiated HL-60 cells. HL-60 cells were cultured in the (A) absence or (B) presence of 100 nM PMA for 48 hr. Fura-2-loaded cells were assayed in a fluorimeter for Ca2+ mobilization in response to the indicated concentrations of UTP. Where indicated, the cells were lysed by the addition of 20 µg/ml digitonin (dig) to permit calibration of the release of Fura-2. C, Concentration-response relationships describing UTP-induced Ca2+ mobilization in (circles) control HL-60 cells or cells treated with 100 nM PMA for () 24 or () 48 hr. D, Concentration-response relationships describing UTP-induced InsP3 production in () control and () 48-hr PMA-treated HL-60 cells. HL-60 cells were labeled with 3H-inositol in serum-free medium as described in Materials and Methods for 24 hr and then cultured in the presence or absence of 100 nM PMA for an additional 48 hr. Cells were washed and resuspended in assay buffer (see Materials and Methods) and incubated with the indicated concentrations of UTP for 15 sec at 37° before the assay for InsP2/InsP3 production.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   UTP-induced Ca2+ mobilization in HL-60 cells after acute or chronic PMA treatment. HL-60 cells were cultured in the presence or absence of 100 nM PMA for 18 hr and Fura-2-loaded as described in the legend to Fig. 1. Top, Ca2+ mobilization in response to 300 µM UTP was measured in control cells, in control cells incubated with 100 nM PMA for 15 min before the assay, and in 18-hr PMA-treated cells. Bottom, UTP-induced Ca2+ mobilization was also measured in the cells treated with 300 nM staurosporine (+Stauro) for 5 min before the assay. After the cells were stimulated with UTP for ~60 sec, 20 µg/ml digitonin was added to release Fura-2 into the extracellular medium for calibration (this causes the steadily maintained increase in fluorescence at the end of each trace). The peak cytosolic [Ca2+] triggered by UTP in each cell suspension is also indicated.

Changes in P_2U receptor mRNA levels during granulocytic versus monocytic differentiation of HL-60 cells. To determine the effects of granulocytic or monocytic differentiating agents on P_2UR mRNA expression, HL-60 cells were treated with Bt₂cAMP or PMA over a time course of 3 days and subjected to Northern blot analysis. These Northern blots were sequentially probed with cDNA probes for P_2UR, myeloperoxidase, IL-1, FPR, and GAPDH. Hybridization with a probe to the carboxyl-terminal half of the human P_2UR coding sequence revealed that a 2.3-kb P_2UR mRNA is abundantly expressed in undifferentiated HL-60 cells (Fig. 3A). P_2UR mRNA levels were transiently up-regulated by 2.5-fold after a 2-hr treatment with Bt₂cAMP; this was followed by a return to preinduction P_2UR mRNA levels after 8 hr and a gradual decline during the following 2 days of in vitro differentiation (Fig. 3A, left top). These relative changes in P_2UR mRNA levels, as quantified by PhosphorImager analysis and normalized to GAPDH mRNA levels, are illustrated in Fig. 3B. We did not determine whether these effects of Bt₂cAMP on P_2UR mRNA levels could be mimicked by physiological agents that increase cAMP. However, previous studies have indicated that most effects of Bt₂cAMP on HL-60 cell differentiation can be elicited when these cells are cotreated with prostaglandin E₂, which activates G_s-coupled prostaglandin receptors, and theophylline, which inhibits phosphodiesterase (17).

View larger version (51K): [in this window] [in a new window]   Fig. 3.   Northern blot analysis of P2UR mRNA levels during differentiation of HL-60 cells by Bt2cAMP or PMA. A, The levels of P2UR mRNA were assayed in HL-60 cells differentiated with 500 µM Bt2cAMP or 100 nM PMA over a 72-hr time course. Poly(A)+ RNA (3.0 µg/lane) was subjected to Northern blot analysis and sequentially probed with 32P-labeled cDNAs corresponding to the human P2UR (top left and right), GAPDH (middle left and right), FPR (bottom left), or IL-1 (bottom right). B, Hybridization signals from the above Northern blots were quantified using a PhosphorImager. The P2UR signals were then normalized to the corresponding GAPDH signal at each time point. Finally, the normalized P2UR signal at each time point was expressed as a ratio relative to the normalized P2UR signal at time zero.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   RT-PCR analysis of P2UR mRNA levels during differentiation of HL-60 cells by Bt2cAMP or PMA. A, P2UR and GAPDH mRNA levels in HL-60 cells differentiated with 500 µM Bt2cAMP over a 72-hr time course. B, P2UR, IL-1, and GAPDH mRNA levels in HL-60 cells differentiated with 100 nM PMA over a 72-hr time course. C and D, Quantification of the amplified P2UR RT-PCR products from the experiments illustrated in A and B, respectively. Each 32P-labeled PCR product band was excised, melted, and analyzed by scintillation counting. Data represent cpm of 32P for each band after subtraction of background radioactivity and are representative of results from three similar experiments.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Concentration-response relationship characterizing the down-regulation of P2UR mRNA by PMA. A, HL-60 cells were treated with the indicated concentrations of PMA for 48 hr, and total RNA was prepared and subjected to semiquantitative RT-PCR analysis using primers for the human P2UR, IL-1, and GAPDH cDNAs. B and C, Quantification of the PCR products was performed as described in the legend to Fig. 4. Data represent cpm of 32P in each PCR product band after correction for background radioactivity.

Stability of P_2UR mRNA in HL-60 cells. The modulation of P_2UR mRNA levels during myeloid differentiation may reflect changes in mRNA stability and/or changes in steady state transcription. To further characterize the regulation of the P_2UR mRNA levels, the stability of P_2UR mRNA and GAPDH mRNA transcripts was assayed by treating HL-60 cells with actinomycin D for 1-4 hr before isolation of total RNA or poly(A)⁺ RNA. Both Northern blot (Fig. 6) and RT-PCR (Fig. 7) analyses indicated that the P_2UR mRNA levels rapidly decreased after the addition of actinomycin. In contrast, no decrease in GAPDH mRNA was observed during the initial 4 hr of actinomycin D treatment. Given the relative stability of the GAPDH mRNA during these actinomycin treatments, P_2UR mRNA levels at each time point were normalized relative to the corresponding GAPDH mRNA levels. These measurements indicated that the P_2UR mRNA half-life was ~60 min (range, 30-75 min in five experiments) in undifferentiated HL-60 cells. This relatively short half-life of the P_2UR mRNA suggests that expression of functional P_2UR can be rapidly altered by changes in transcription or mRNA stability. Other experiments tested whether the stability of P_2UR mRNA was acutely altered by Bt₂cAMP or PMA, the agents used to induce in vitro differentiation along the granulocytic or monocytic pathways (Fig. 7). RT-PCR analysis indicated that the ~60-min half-life of the P_2UR mRNA transcripts was not significantly affected by either agent during the 4-hr treatment with actinomycin D. 

View larger version (40K): [in this window] [in a new window]   Fig. 6.   Half-life of P2UR mRNA in undifferentiated HL-60 cells. HL-60 cells were treated with 5 µg/ml actinomycin D for the indicated times. Poly(A)+ RNA was subjected to Northern blot analysis, which was performed and quantified as described in the legend to Fig. 3. Data represent the changes in P2UR mRNA level relative to time zero after normalization to the corresponding GAPDH mRNA level at each time point and are representative of duplicate experiments.

View larger version (36K): [in this window] [in a new window]   Fig. 7.   RT-PCR analysis of P2UR mRNA half-life in control HL-60 cells and HL-60 cells acutely treated with Bt2cAMP or PMA. A, HL-60 cells were treated with 5 µg/ml actinomycin D (ActD) alone (left) or in the additional presence of 500 µM Bt2AMP (middle) or 100 nM PMA (right) for the indicated times. Total RNA was prepared and subjected to semiquantitative RT-PCR analysis using primers to the human P2UR and GAPDH as described in the legend to Fig. 4. B and C, Quantification of the P2UR and GAPDH PCR products was performed as described in the legend to Fig. 4. These values are representative of results from two experiments.

Effects of PMA on P_2UR mRNA levels in other myeloid and nonmyeloid cell types. P_2UR expression was assayed during the PMA-induced differentiation of THP-1 monocytes, another human myeloid leukocyte line. Unlike the pluripotent HL-60 line, THP-1 cells are irreversibly committed to the monocyte/macrophage lineage (20). P_2UR mRNA levels were reduced by 10-fold in THP-1 cells treated with 100 nM PMA for 1 or 2 days (Fig. 8A). Like in HL-60 cells, these changes in P_2UR transcript levels were inversely correlated with the up-regulation of IL-1 mRNA. After 3 days of induction with PMA, the level of P_2UR mRNA increased slightly, whereas the amount of IL-1 mRNA was markedly reduced.

View larger version (19K): [in this window] [in a new window]   Fig. 8.   Differential effects of chronic PMA treatment on the down-regulation of P2UR mRNA in myeloid and nonmyeloid cells. A, THP-1 promonocytes were treated with 100 nM PMA for 0, 24, or 48 hr. RNA, isolated from each cell sample, was analyzed by RT-PCR using primers for the human P2UR, IL-1, and GAPDH cDNAs. B, A431 human epidermal cells were treated with 100 nM PMA for 0, 12, 24, or 48 hr; human keratinocytes were treated with 100 nM PMA 0, 24, or 48 hr. RNA, isolated from each sample, was analyzed by RT-PCR using primers to the human P2UR and GAPDH cDNAs. Only the P2UR products are shown.

Down-regulation of P_2UR mRNA expression during inflammatory activation of myeloid leukocytes. In addition to inducing HL-60 promyelocytes and THP-1 monocytes to differentiate along the monocyte/macrophage pathway, PMA modulates the expression of many genes associated with inflammatory activation of mature monocyte/macrophages (13, 20, 21). Several observations suggested that the marked down-regulation of P_2UR expression in PMA-treated HL-60 cells may be indicative of inflammatory activation rather than simple commitment to monocyte/macrophage differentiation. First, freshly isolated blood monocytesexhibit robust Ca²⁺-mobilizing responses to micromolar ATP and UTP (9). Second, the PMA-induced down-regulation of P_2UR mRNA in both HL-60 cells and THP-1 cells correlates with the up-regulation of proinflammatory cytokines, such as IL-1 (Figs. 3, 4, 5 and 8) and TNF- (data not shown). To further address this possibility, we tested whether physiological inflammatory agents, such as IFN- and bacterial endotoxin/LPS, might also induce down-regulation of P_2UR expression in THP-1 cells that are already committed to the monocyte/macrophage lineage. It should be noted that IFN- induces some but not all phenotypic changes that characterize inflammatory activation of mononuclear phagocytes (13, 22). Likewise, although LPS alone can induce most phenotypic changes that characterize inflammatory activation of macrophages, the rate of such induction by LPS can be greatly potentiated when monocyte/macrophages are simultaneously exposed to IFN- (13). Fig. 9A shows that IFN- alone induced no changes in P_2UR expression at 24 hr and only a minor decrease at 48 hr. Consistent with its being a priming agent rather than a full inflammatory activator, IFN- induced a delayed up-regulation of TNF- (particularly evident at 48 hr) but no increase in IL-1 mRNA. In contrast, cotreatment of THP-1 cells with both IFN- and LPS greatly up-regulated the expression of TNF- and IL-1 within 24 hr. This up-regulation of inflammatory cytokines was correlated with a ~5-fold decrease in P_2UR mRNA levels. UTP-induced Ca²⁺ mobilization was also significantly decreased in THP-1 cells treated with both LPS and IFN- (data not shown). The ability of IFN- to prime THP-1 cells for the down-regulation of P_2UR mRNA by either LPS or PMA was further characterized by pretreating these cells with interferon for 48 hr before relatively short (8-hr) exposures to either PMA or LPS (Fig. 9B). We also tested the potential priming effects of 1,25-dihydroxy-vitamin D₃, which can induce monocytic differentiation but not inflammatory activation of human myeloid cell lines (20). Treatment of nonprimed THP-1 cells with LPS or PMA for 8 hr did not change the intensities of the P_2UR RT-PCR signals (as visually indicated by ethidium fluorescence). In cells primed with 1,25-dihydroxy-vitamin D₃, an 8-hr exposure to PMA but not LPS reduced the intensity of the P_2UR RT-PCR signal relative to that observed in the untreated cells. In contrast, the short term exposure to either LPS and PMA induced a more significant down-regulation of P_2UR mRNA in THP-1 cells primed with IFN- for 48 hr.

View larger version (34K): [in this window] [in a new window]   Fig. 9.   Down-regulation of P2UR mRNA during inflammatory activation of THP-1 monocytes with IFN- and endotoxin/LPS. A, THP-1 monocytes were treated for 24 or 48 hr with 103 units/ml IFN- alone (Ifn) or with 103 units/ml IFN- plus 1 µg/ml endotoxin/LPS (Ifn/LPS). RNA from these treated cells and from control, untreated THP-1 monocytes (Con) was analyzed by RT-PCR using primers for P2UR, IL-1, TNF-, and GAPDH. A parallel sample of RNA from the control THP-1 cells was subjected to the same RT-PCR protocol in the absence of RT enzyme (mock). B, THP-1 monocytes were cultured for 48 hr (priming incubation) with no priming agents (None), with 10 ng/ml 1,25-dihydroxy-vitamin D3 (Vit D3), or with 1000 µ/ml IFN- (-Ifn). Each suspension of primed cells was divided into three aliquots that were cultured for an additional 8 hr with no inducing agent (Con), with 1 µg/ml LPS, or with 100 nM PMA. RNA, isolated from each group of primed/induced cells, was analyzed by RT-PCR using primers for human P2UR cDNA and GAPDH cDNA. Only the P2UR PCR products are shown. Parallel samples of RNA from the control and -IFN-treated cells were subjected to mock RT-PCR analysis (mock).

	Discussion

Summary Introduction Materials & Methods Results Discussion References

Neutrophils and monocytes express P_2U-purinergic receptors that mobilize intracellular Ca²⁺ in response to extracellular ATP or UTP (9, 10). Although cDNAs encoding this G protein-coupled receptor have been isolated from several species and tissue sources (2), the promoter sequence of the human P_2UR gene has not been characterized, and little is known regarding the factors that regulate expression of this receptor. Because traditional methods for studying receptor number at the cell surface, such as ligand or antibody binding, are unavailable for most ATP receptors, we studied the regulation of P_2UR expression at the mRNA level. We demonstrate that the expression of the P_2UR mRNA in human myeloid leukocytes is regulated by both agents that induce differentiation of myeloid progenitor cells and agents that trigger inflammatory activation of these leukocytes. This is the first evidence for plasticity in the expression of P_2UR during defined programs of cellular differentiation.

Bt₂cAMP induces HL-60 cells to acquire morphological characteristics of granulocytes/neutrophils (14, 17, 23). We have previously reported that the potency and efficacy of ATP as a Ca²⁺-mobilizing agonist were virtually identical in undifferentiated HL-60 promyelocytes and in HL-60 granulocytes (4). Although P_2UR mRNA levels always decreased during prolonged treatment of HL-60 cells with Bt₂cAMP, the steady state levels after 2 days of induction were usually only 2-3-fold lower than that in uninduced cells (Figs. 3 and 4). P_2UR mRNA was also expressed at approximately similar levels in HL-60 granulocytes and freshly isolated human neutrophils (data not shown). The relatively modest effect of a granulocytic-inducing agent, such as Bt₂cAMP, on the level of P_2UR mRNA in HL-60 cells is consistent with the maintained expression of functional P_2UR in both HL-60 granulocytes and human blood neutrophils. The reduction in P_2UR mRNA was preceded by a transient increase in P_2UR transcript levels, which peaked within 2 hr (Figs. 3 and 4). This transient increase was also observed in PMA-treated cells and presumably involved enhanced transcription because no increase was observed in the presence of actinomycin D (Fig. 8). The physiological significance of this transient increase is unclear, and it remains to be determined whether these effects can be mimicked by receptor agonists that elevate cAMP. However, Collins et al. (24) observed a similar, rapid increase in ₂-adrenergic receptor mRNA, followed by a modest decrease in DDT1-MF2 smooth cells treated with either Bt₂cAMP or epinephrine, a physiological agonist for ₂-adrenergic receptor. A cAMP-response element present in the promoter of ₂-adrenergic receptor gene was implicated in the up-regulation of that receptor mRNA. The very similar, triphasic changes in P_2UR or ₂-adrenergic receptor mRNA induced by Bt₂cAMP suggests that a cAMP-response element may be present in the human P_2UR gene promoter.

HL-60 cells treated with PMA acquire many of the morphological and functional characteristics of monocytes and macrophages (14, 21). These PMA-differentiated cells were largely unresponsive to even maximally activating concentrations of UTP in both Ca²⁺ mobilization and InsP₃ production assays (Figs. 1 and 2). This lack of P_2UR functional activity correlated with a significant down-regulation of P_2UR mRNA to values ~10-fold lower than that measured in undifferentiated cells (Figs. 3, 4, 5). The correlation between the loss of P_2UR function in cell membranes (Table 1) and the reduction in P_2UR mRNA levels suggests that PMA-induced down-regulation of these receptors primarily reflects decreased expression of functional receptor protein at the cell surface. Although P_2UR mRNA levels were down-regulated in both Bt₂cAMP- and PMA-differentiated HL-60 cells, a significant attenuation of P_2UR functional activity (as indicated by the agonistic potencies and efficacies of UTP or ATP) was observed only in the PMA-treated HL-60 cells. It is possible that chronic PMA treatment also increases the internalization and degradation of P_2UR. A PMA-induced increase in P_2UR internalization/degradation in combination with the large reduction in steady state P_2UR mRNA levels may lead to the dramatic loss of P_2UR function that was observed in PMA-differentiated HL-60 cells but not in the Bt₂cAMP-induced cells (4). It should also be noted that phorbol esters have been shown to induce a profound down-regulation of other G protein-coupled receptors at both mRNA and protein levels; these include the ₃-adrenergic receptor in adipocytes (25), the M2-muscarinic receptor in lung cells (26), and the thrombin receptor in mesangial cells (27).

Inflammatory activation constitutes a second stage of phagocyte development, distal to the primary commitment to monocytic or granulocytic differentiation. PMA is a potent inducer of the inflammatory phenotype (13, 20). For example, the mRNA for IL-1, a major proinflammatory cytokine, was strongly induced during PMA treatment of HL-60 promyelocytes (Figs. 3 and 4) and THP-1 monocytes (Fig. 8A). This suggested that PMA-induced down-regulation of P_2UR expression might be mimicked by physiological activators of inflammation. Consistent with this possibility, we observed that cotreatment of THP-1 monocytes with IFN- and LPS strongly down-regulated P_2UR mRNA levels (Fig. 9). Therefore, the profound down-regulation of P_2UR mRNA and functional P_2UR observed in PMA-treated myeloid leukocyte may be associated with the second, inflammatory stage of differentiation rather than the primary differentiation to the monocytic phenotype. It remains to determined whether down-regulation of the P_2UR mRNA by chronic PMA treatment is an exclusively myeloid-specific phenomenon. The absence of significant P_2UR mRNA down-regulation by PMA in A431 epithelial carcinoma cells and human keratinocytes (Fig. 8B) indicates that down-regulation of P_2UR mRNA is not a generalized response to PKC activation. The strong temporal correlation between down-regulation of P_2UR expression and the up-regulation of IL-1 expression in PMA-induced HL-60 cells raises the possibility that P_2UR down-regulation also involves autocrine input from proinflammatory cytokines, such as IL-1 and TNF-.

Another significant finding was that the P_2UR transcript is relatively short lived (t_1/2 = 60-90 min) in both myeloid and nonmyeloid cells. A short half-life may facilitate rapid modulation of P_2UR expression in different functional or developmental states of both myeloid and nonmyeloid tissues. The mechanism underlying this rapid turnover of the P_2UR mRNA remains to be determined. The half-life of the P_2UR mRNA was unchanged during 4-hr treatments of HL-60 cells with Bt₂AMP or PMA (Fig. 7). This indicates that down-regulation of P_2UR mRNA levels cannot be due simply to acute changes in mRNA stability triggered by PKC- or PKA-dependent phosphorylation of pre-existing RNA stability factors. However, this does not rule out a delayed effect on P_2UR mRNA stability in cells treated for prolonged times (>8 hr) with PMA or Bt₂cAMP. A delayed effect could indicate a requirement for de novo synthesis of a factor that further decreases the stability of P_2UR mRNA.

The ability of phorbol esters to down-regulate P_2UR expression in myeloid leukocytes raises the question of whether a similar down-regulation might be elicited by physiological agents that activate diglyceride accumulation and PKC-based signaling. Other than P_2UR, undifferentiated HL-60 cells do not express most of the PI-PLC-coupled receptor types present in mature myeloid leukocytes (e.g., the receptors for formyl peptides, platelet activating factor, or leukotriene B; for a review, see Ref. 27). We previously reported that chronic treatment (5 days) of HL-60 cells with adenosine-5-O-(3-thio)triphosphate (added every 12 hr to offset breakdown) induces a partial down-regulation of P_2UR, as assayed by reduced Ca²⁺ mobilization in response to ATP (28). Preliminary RT-PCR analyses also suggest that P_2UR mRNA levels are reduced by ~50% in HL-60 cells treated with 100 µM ATP every 6-8 hr for 2 days. Further experiments are required to verify this autocrine down-regulation. However, such results may be similar to the findings of Chau et al. (29), who reported an autocrine down-regulation of platelet activating factor receptor mRNA in U937 human promonocytes (a related human myeloid line) chronically stimulated with a poorly metabolizable platelet-activating factor analog.

The PI-PLC signaling pathway in mature phagocytic leukocytes is rapidly activated by many inflammatory agonists and is involved in the regulation of chemotaxis, secretion, phagocytosis, and superoxide release. The activation of P_2UR in mature human neutrophils and monocytes primes these cells for enhanced superoxide release in response to other inflammatory agonists, such as formyl peptides (10, 11). ATP or UTP stimulation of the P_2UR also increases neutrophil adherence to endothelial cells (30, 31) and the activation of CD11b/CD18 integrins in neutrophils (32). Recent studies have verified that ATP and UTP are very effective chemotactic stimuli for HL-60 granulocytes and human neutrophils (33). Our data suggest that P_2UR are primarily expressed in the marrow-restricted myeloid progenitor cells and blood-borne neutrophils and monocytes. Like other G protein-coupled chemoattractant receptors (34), P_2UR may play a role in the recruitment of blood neutrophils and monocytes to sites of tissue inflammation. Once neutrophils or monocytes have entered these sites, the continued expression of primarily chemotactic receptors may be counterproductive and subject to down-regulation by inflammatory cytokines. Lloyd et al. (35) reported that expression of IL-8 receptor mRNA and protein is markedly reduced in neutrophils treated with LPS or TNF-. This is similar to the down-regulation of P_2UR mRNA observed in THP-1 monocytes treated with LPS and IFN-. It will be interesting to determine whether P_2UR are down-regulated in other myeloid cell types, such as neutrophils, and in nonmyeloid cells, such as endothelial cells, which exhibit rapid changes in gene expression in response to LPS, TNF-, and IL-1.