Cyclic AMP induces activation of extracellular signal-regulated kinases in HL-60 cells: role in cAMP-induced differentiation

2.1. Reagents 

8Br-cAMP was purchased from Boehringer Mannheim (Indianapolis, IN); PD98059 and forskolin were from Calbiochem-Novabiochem (San Diego, CA); anti-phosphorylated ERK, anti-ERK, and anti-MEK antibodies were from New England BioLabs (Beverly, MA); anti-B-Raf antibody (#06-532) and inactive MEK-1-GST for the kinase assay were from Upstate Biotechnology (Lake Placid, NY); anti-B-Raf antibody for Western blotting was from Santa Cruz Biotechnology (Santa Cruz, CA); luminol and TPA were purchased from Sigma (St. Louis, MO). All other reagents were of the highest purity available.

2.2. Cell culture and induction of differentiation 

HL-60 cells were purchased from the American Type Culture Collection (Rockville, MD). Cells were maintained in RPMI 1640 medium supplemented with 15% fetal bovine serum (FBS; HyClone, Logan, UT). In experiments, cells growing logarithmically were serum-starved for 24h in medium containing 0.5% FBS. To induce differentiation, cells in logarithmic phase were seeded at 5×105/ml and treated with 1mM 8Br-cAMP for 48h. To test the role of ERK activation in differentiation, 20μM PD98059 was added to the culture 30min before treatment with 8Br-cAMP.

2.3. Western blot analysis 

Cells were harvested and lysed in buffer containing 25mM HEPES (pH 8.0), 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 137mM NaCl, 2mM EDTA, 1mM Na3VO4, 1mM NaF, 1mM phenylmethylsulfonyl fluoride, 5μg/ml aprotinin, and 5μg/ml leupeptin. Insoluble materials were removed by centrifugation for 10min at 15,000×g. Protein concentrations were measured by BCA protein assay reagent (Pierce, Rockford, IL). Proteins (40mg) were separated by 9% SDS–polyacrylamide gel electrophoresis (SDS–PAGE) under reducing conditions, and Western blotting was performed using the methods of Choi et al. [12]. Immune complexes were detected by chemiluminescence (SuperSignal WestDura, Pierce) under the conditions recommended by the supplier.

2.4. Immunoprecipitation and B-Raf kinase assay 

B-Raf kinase was assayed with the B-Raf Immunoprecipitation Kinase Cascade Assay Kit (Upstate Biotechnology) following the manufacturer’s protocol. B-Raf was immunoprecipitated using anti-B-Raf antibody and protein G–agarose beads. The beads were washed, then incubated with 1μg recombinant MEK-1 and 10μCi γ-[] ATP in assay buffer (20mM MOPS (pH 7.2), 25mM β-glycerophosphate, 5mM EGTA, 1mM Na3VO4, 1mM dithiothreitol) for 30min at 30°C. Reaction products were separated by SDS–PAGE, and the dried gel exposed to X-ray film or analyzed with a BioImaging BAS2500 Analyzer (Fuji, Tokyo, Japan).

2.5. Chemiluminescence assay 

Superoxide production by HL-60 cells was measured by luminol-enhanced chemiluminescence and recorded with a luminometer (Bio-Rad, Hercules, CA), using the methods of Cho et al. [13]. Cells were washed twice with Dulbecco’s phosphate-buffered saline (PBS, 137mM NaCl, 2.7mM KCl, 8mM Na2HPO4, 1.5mM KH2PO4, 0.9mM CaCl2, 0.49mM MgCl2, 5.6mM d-glucose, 0.33mM sodium pyruvate) containing 0.1% bovine serum albumin and resuspended at 2×106 cells per ml. A 300μl aliquot of cells and 100μl PBS containing luminol (100μM) were added to the cuvettes of the luminometer and allowed to equilibrate for 5min at 37°C before stimulation with TPA (final concentration, 3μM). The intensity of the developing chemiluminescence was measured for 30min at 37°C and the results were presented graphically with time units on the abscissa and light intensity units (mV) on the ordinate. The values (in V) for peak height per 1×105 viable cells were evaluated. Data are presented as mean±standard deviation (S.D.). Statistical significance was determined by Student’s t-test.

2.6. FACS analysis for CD14 expression 

CD14 expression was measured using a fluorescence-activated cell sorter (FACS) with the methods of Cho et al. [13]. Cells (1×106) resuspended in PBS containing 0.1% sodium azide and 5% FBS were incubated at 4°C for 30min with 1μg FITC-conjugated anti-CD14 antibody (BioSource, Camarillo, CA). The cells were then washed twice and fixed with 2% formaldehyde in PBS. Fluorescence was analyzed with a FACSVantage flow cytometer using Cellquest software (Becton Dickinson, Bedford, MA).

3.1. Cyclic AMP induces ERK activation 

To assess the involvement of ERK activation in the cAMP-induced differentiation of HL-60 cells, we analyzed the phosphorylation state of ERK 30min after the addition of various concentrations of 8Br-cAMP. An antibody specific for the doubly-phosphorylated form of ERK was used to probe the Western blot (Fig. 1A, upper half), and the equivalence of protein per lane was demonstrated with an antibody that recognizes ERK-2 irrespective of its phosphorylation state (Fig. 1A, lower half). The phosphorylation of endogenous ERK in HL-60 cells increased in a dose-dependent manner, up to 3mM 8Br-cAMP. A time-course analysis showed that cAMP-induced the rapid activation of ERK within 5min, and this was sustained for up to 6h (Fig. 1B). To confirm that the activation of ERK was cAMP-mediated, we tested the effects of another intracellular cAMP-elevating agent, forskolin, which stimulates adenylyl cyclase. Consistent with the findings after 8Br-cAMP treatment, 10μM forskolin stimulated ERK phosphorylation (Fig. 1C).

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Fig. 1. 8Br-cAMP induces the activation of ERK in HL-60 cells. (A) Cells were cultured in medium containing 0.5% FBS for 24h before addition of the indicated concentrations of 8Br-cAMP for 30min. The cell lysates were analyzed by Western blot using either an antibody specific for active ERK-1 and ERK-2 doubly-phosphorylated at Thr202/Tyr204 (upper panel), or with anti-ERK antibody to control for protein loading (lower panel). (B) The kinetics of cAMP-induced ERK phosphorylation were observed by incubating the cells with 1mM 8Br-cAMP for the periods indicated (0–6h). (C) The effect of 20μM forskolin on the phosphorylation of ERKs for the periods indicated (0–1h).

3.2. Upstream components of the ERK signaling pathway 

To identify the upstream signal, we next examined the activation of MEK 30min after the addition of 8Br-cAMP. The phosphorylation of MEK increased in a dose-dependent manner with 8Br-cAMP treatment (Fig. 2A). Moreover, PD98059, a MEK-1 inhibitor, completely blocked ERK phosphorylation, indicating that ERK activation is mediated by MEK (Fig. 2B).

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Fig. 2. 8Br-cAMP-induced activation of ERK is mediated by MEK in HL-60 cells. (A) Dose-dependent activation of MEK by 8Br-cAMP in HL-60 cells. Quiescent cells were treated for 30min with the concentrations of 8Br-cAMP indicated. The blots were probed with an antibody specific for active MEK, phosphorylated at Ser217/221 (upper panel), and anti-MEK antibody to control for loading (lower panel). (B) Effect of MEK-1 inhibitor on the 8Br-cAMP-induced activation of ERK. Quiescent cells were treated with 10μM PD98059 for 30min before 1mM 8Br-cAMP was added for 30min. The blots were probed with an antibody specific for phosphorylated ERK (upper panel), or with anti-ERK antibody to control for protein loading (lower panel).

In B-Raf-expressing cells, ERK activation by cAMP has been observed and has been attributed to Rap1 activation, with the subsequent activation of B-Raf. Previous studies have suggested that Rap1 may be important in the differentiation of HL-60 cells [14], [15], [16]. For example, cGMP induces the activation of Rap1 in parental HL-60 cells, but not in variant cells which are resistant to cGMP-induced differentiation [17]. Therefore, we examined whether B-Raf is the molecule upstream from MEK activation. In contrast to the finding that cAMP activates MEK and ERK, B-Raf activity was inhibited by 8Br-cAMP (Fig. 3A). Pretreatment with H89, a PKA inhibitor, blocked the cAMP-mediated inhibition of B-Raf (Fig. 3B). These results indicate that B-Raf activity is negatively regulated by cAMP via a PKA-dependent mechanism.

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Fig. 3. 8Br-cAMP inhibits B-Raf kinase activity in HL-60 cells. (A) The negative regulation of B-Raf kinase by 8Br-cAMP. Quiescent cells were treated with 1mM 8Br-cAMP for the periods indicated (0–6h). Cell lysates were immunoprecipitated using a B-Raf-specific antibody that recognizes residues 10–22 of human B-Raf kinase. Immunoprecipitates were incubated in the presence of MEK-1 and γ-[] ATP for 30min and subjected to SDS–PAGE and autoradiography to assess MEK phosphorylation (upper panel). The lysates were analyzed in parallel with anti-B-Raf antibody (lower panel). (B) Effect of PKA inhibitor on the inhibition of B-Raf kinase by 8Br-cAMP. H89 (10μM) was added to cell cultures 30min before treatment with 1mM 8Br-cAMP for 30min. Cell lysates were immunoprecipitated and assayed for B-Raf kinase protein, as described.

3.3. cAMP-induced ERK activation induces the differentiation of HL-60 cells 

We next investigated whether cAMP-mediated ERK activation plays an essential role in the process of differentiation. The functional phenotype of differentiated HL-60 cells was identified by their inducible oxidative metabolism, a characteristic of mature myelomonocytic cells. Cells cultured in the presence of 8Br-cAMP for 48h generated 4.5-fold more TPA-stimulated superoxide than cells cultured in the absence of 8Br-cAMP. This increase was significantly inhibited, to 1.7-fold, by pretreatment with PD98059 (Fig. 4). Flow cytometric analysis of CD14 expression demonstrated that the proportion of CD14 antigen-positive cells increased from 4.2 to 63.5% when cultured in the presence of 8Br-cAMP (Fig. 5). Moreover, the cAMP-induced expression of CD14 antigen was inhibited from 63.5 to 35.0% by treatment with PD98059 (Fig. 5). These results clearly show that cAMP-induced ERK activation plays a role in the differentiation of HL-60 cells.

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Fig. 4. TPA stimulates superoxide generation in HL-60 cells pretreated with 8Br-cAMP. Cells cultured in medium containing 1mM 8Br-cAMP with or without 20μM PD98059 for 2 days were stimulated with 3μM TPA. Maximal generation of luminol-enhanced chemiluminescence was measured as described in Section 2. The data are presented as the mean±S.D. of four independent experiments; ∗ P<0.05, compared with cells treated with 8Br-cAMP only.

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Fig. 5. 8Br-cAMP induces CD14 expression in HL-60 cells. Cells were cultured in medium containing 1mM 8Br-cAMP with or without 20μM PD98059 for 2 days. The cells were probed with FITC-conjugated anti-CD14 antibody, and the fluorescence was analyzed as described in Section 2. Percentages of cells positive for CD14 are indicated.