Abstract

Previous studies have demonstrated that the activity of GM3 synthase and GM3 content are increased during the differentiation of human promyelocytic leukemia HL-60 cells into the monocyte/macrophage lineage after phorbol 12-myristate 13-acetate (PMA) treatment. However, the molecular mechanisms involved in transcriptional activation of GM3 synthase during differentiation of PMA-induced HL-60 cells are not well understood. As evidenced by western blot analysis, PMA induced the marked activation of protein kinase C (PKC)/extracellular regulated kinases (ERKs) signal transduction pathway during the differentiation of HL-60 cells. In addition, PKC/ERKs activation induced by PMA in HL-60 cells led to the phosphorylation of cAMP-responsive element binding protein (CREB) as a transcription factor. In PMA-stimulated HL-60 cells, the PKC/ERKs-dependent CREB activation regulated expression of GM3 synthase, inducing a synthesis of ganglioside GM3 product. On the other hand, although ganglioside GM3 was shown to be able to induce the differentiation of HL-60 cells into the monocyte/macrophage lineage, effect of ganglioside GM3 on expression of CD11b as a differentiation marker in HL-60 cells has been not reported yet. Interestingly, the increased ganglioside GM3 through PKC/ERKs/CREB-dependent pathway by PMA resulted in an increase of CD11b surface antigen expression and induction of HL-60 cells adherence. Treatment with L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), glucosylceramide synthase inhibitor, decreased induction of not only CD11b expression but also cellular adherence by reduction of PMA-induced ganglioside GM3. Furthermore, treatment of HL-60 cells with exogenous ganglioside GM3 induced CD11b expression. These results show that the enhanced expression of GM3 synthase through PKC/ERKs-dependent CREB activation by PMA is associated with the differentiation of HL-60 cells by inducing expression of CD11b known as a monocyte/macrophage differentiation maker.

Introduction

Gangliosides are found on the outer leaflet of the plasma membrane of vertebrate cells and are particularly abundant in the central nervous system (Svennerholm, 1980). They are sialic acid–containing glycosphingolipids that participate in cellular proliferation, differentiation, adhesion, signal transduction, tumorigenesis, and metastasis (Birkle et al., 2003; Varki, 1993). Ganglioside GM3 was able to regulate fibroblast growth factor and epidermal growth factor receptor activities through the modulation of tyrosine-kinase activity (Bremer and Hakomori, 1982; Bremer et al., 1986). GM3 is also able to regulate the adhesion and migration of several carcinoma and other cell lines grown on fibronectin (Iwabuchi et al., 1998; Kawakami et al., 2002; Ono et al., 2001; Satoh et al., 2001; Watanabe et al., 2002; Zheng et al., 1992, 1993). Recently, GM3, in addition to its suppression of cell motility, was also shown to inhibit tumor cell invasion (Kawamura et al., 2001; Watanabe et al., 2002). When the human promyelocytic leukemia cell line (HL-60) is induced to monocyte/macrophage differentiation by phorbol 12-myristate13-acetate (PMA), the activity of CMP-NeuAc:lactosylceramide sialyltransferase (GM3 synthase) increased (Momoi et al., 1986) and its product, ganglioside GM3, was increased (Momoi and Yokota, 1983; Momoi et al., 1986; Nojiri et al., 1984). In addition, the exogenous treatment with ganglioside GM3 induces the differentiation of HL-60 cells into monocytic lineage or macrophage-like cells (Nojiri et al., 1986; Stevens et al., 1989).

HL-60 cells can be induced into the monocyte/macrophage or granulocyte pathways by exposure to various chemical reagents (Breitman et al., 1980; Collins et al., 1978; Rovera et al., 1979). Dimethyl sulfoxide or retinoic acid induce the differentiation of HL-60 cells into the granulocyte lineage (Breitman et al., 1980; Collins et al., 1978), whereas PMA causes the differentiation of monocyte/macrophage in HL-60 cells (Rovera et al., 1979). PMA-induced differentiation of a number of leukemia cell lines including HL-60 cells has been extensively investigated. The effects of PMA are due to the activation of protein kinase C (PKC)-b (Macfarlane and Manzel, 1994; Tonetti et al., 1994) and resulted in rapid and sustained activation of extracellular regulated kinase (ERK) 1 and ERK2 in differentiation of HL-60 cells (Das et al., 2000; Miranda et al., 2002). Additionally, induction of terminal differentiation in leukemia cells by PMA is also associated with the activation of the stress-activated protein kinase, release of cytochrome c, activation of caspase, induction of p21WAF and p27kip1, down-regulation of bcl-2, and ultimately the induction of apoptosis (Das et al., 2000; Ito et al., 2001; Laouar et al., 2001; Sakakura et al., 1996). However, the molecular mechanisms involved in transcriptional activation of GM3 synthase during differentiation of PMA-induced HL-60 cells are not well understood, although GM3 synthase activity and ganglioside GM3 product are increased during HL-60 cells differentiation.

The CD11b (macrophage-1 antigen) leukocyte integrin (α) subunit exists on the surface of granulocytes and monocyte/macrophages coupled with CD18 (β) subunit in an α1/β1 heterodimer. This heterodimer mediates leukocyte adherence activity of these myeloid cells (Hickstein et al., 1992). When several chemicals induced HL-60 cell differentiation to granulocytes and PMA promoted HL-60 cell differentiation to macrophages, these cells increased the expression of CD11b, and the CD11b/CD18 complex was located on surface of these cells (Collins et al., 1977; Hickstein et al., 1989). However, the relationship between expression of CD11b as a differentiation marker and increase of GM3 during differentiation of PMA-induced HL-60 cells is not fully elucidated.

In this study, we investigated the molecular mechanism for PKC/ERKs-dependent activation of cAMP-responsive element binding protein (CREB) during the differentiation of HL-60 cells induced by PMA and whether expression of GM3 synthase, resulting in an increase in the content of GM3 during the differentiation of HL-60 cells, increases through their signal pathways. Recently, we isolated the promoter of human GM3 synthase from human fetal brain, and this gene was functionally characterized in human hepatoma HepG2 and neuroblastoma SK-N-MC cells (Kim et al., 2002). To further investigate molecular mechanism of GM3 synthase expression during the differentiation of HL-60 cells induced by PMA, we also examined whether the expression of GM3 synthase during HL-60 cell differentiation increased through CREB motif in promoter of GM3 synthase using the luciferase reporter assay and the electrophoretic mobility shift assay (EMSA). In addition, we examined whether PMA-induced ganglioside GM3 in HL-60 cells and exogenous GM3 affected the transcriptional activity of CD11b. We have shown for the first time that the up-regulation of GM3 synthase through PKC/ERKs-dependent CREB activation results in the differentiation of HL-60 cells by inducing expression of CD11b.

Results

Increased transcriptional activation of GM3 synthase and the content of ganglioside GM3 in PMA-stimulated HL-60 cells

We examined the expression of GM3 synthase and its product, ganglioside GM3, as regulated during the differentiation of PMA-induced HL-60 cells. As shown in Figure 1A and B, mRNA levels of GM3 synthase were increased in HL-60 cells induced by PMA, as evidenced by northern blot analysis and reverse transcription polymerase chain reaction (RT-PCR). The mRNA expression of GM3 synthase was detected at 12 h after PMA treatment and increased up to 48 h. In addition, as shown in Figure 1C, ganglioside GM3 was observed during the differentiation of HL-60 cells induced by PMA, as evidenced by high-performance thin-layer chromatography (HPTLC) immunostaining. Furthermore, we first investigated if the promoter activity of GM3 synthase was stimulated during HL-60 differentiation by PMA treatment.

Fig. 1.

Transcriptional activation of GM3 synthase and the increased ganglioside GM3 in HL-60 cells after PMA treatment. Total RNA from HL-60 cells was isolated after 0, 6, 12, 24, or 48 h of PMA treatment and GM3 synthase mRNA was detected by northern blot analysis (A) and RT-PCR (B). Beta-actin was included as an internal control. Glycolipids from HL-60 cells were isolated after 0, 6, 12, 24, or 48 h of PMA treatment. After separation of gangliosides on HPTLC, gangioside GM3 was detected by HPTLC immunostaining with M2590 antibody (C). HL-60 cells were transiently transfected with pGL3-1600, which contains 1600 bp of GM3 synthase promoter, and then cultured with or without PMA for 24 h. Luciferase activity was determined from each cell lysates as described under Materials and methods. The results shown are means ± SD of three independent experiments with triplicate measurement (D).

Fig. 1.

Transcriptional activation of GM3 synthase and the increased ganglioside GM3 in HL-60 cells after PMA treatment. Total RNA from HL-60 cells was isolated after 0, 6, 12, 24, or 48 h of PMA treatment and GM3 synthase mRNA was detected by northern blot analysis (A) and RT-PCR (B). Beta-actin was included as an internal control. Glycolipids from HL-60 cells were isolated after 0, 6, 12, 24, or 48 h of PMA treatment. After separation of gangliosides on HPTLC, gangioside GM3 was detected by HPTLC immunostaining with M2590 antibody (C). HL-60 cells were transiently transfected with pGL3-1600, which contains 1600 bp of GM3 synthase promoter, and then cultured with or without PMA for 24 h. Luciferase activity was determined from each cell lysates as described under Materials and methods. The results shown are means ± SD of three independent experiments with triplicate measurement (D).

GM3 synthase promoter (pGL3-1600) and pGL-3-basic plasmid were transfected into HL-60 cells with or without PMA, respectively, and luciferase activity was measured using the luciferase reporter assay system. As shown in Figure 1D, luciferase activity remained essentially unchanged when the pGL3-basic was transfected into HL-60 cells treated with or without PMA and the pGL3-1600 was transfected into HL-60 cells treated without PMA. However, the transcriptional activity of pGL3-1600 in HL-60 cells with PMA treatment was significantly higher than that in HL-60 cells without PMA treatment. These results clearly show that the expression of both GM3 synthase and ganglioside GM3 are increased during differentiation of HL-60 cells induced by PMA.

Regulation of GM3 synthase via PKC/ERKs pathway but not PI-3K, p38 MAPK, and JNKs pathways in HL-60 cells induced by PMA

We first investigated the signal transduction pathway that leads to the expression of GM3 synthase in the induction of HL-60 differentiation by PMA. As shown in Figure 2A, northern blots and RT-PCR showed that expression of GM3 synthase was increased in PMA-stimulated HL-60 cells, compared with untreated HL-60 cells. However, both PKC (GÖ6976) and ERKs (U0126) inhibitors resulted in a decrease of GM3 synthase expression in HL-60 cells induced by PMA. On the other hand, enhanced expression of GM3 synthase in PMA-stimulated HL-60 cells was not inhibited by phosphatidylinositol-3 kinase (PI-3K), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinases (JNKs) inhibitors (wortmannin, SB203580, and SP600125), respectively, compared with HL-60 cells induced by PMA in absence of chemical inhibitors, as evidenced by northen blot analysis and RT-PCR. This result suggests that transcriptional activation of GM3 synthase in PMA-differentiated HL-60 cells is related to PKC and ERKs pathways.

Fig. 2.

The enhanced expression of GM3 synthase through PKC/ERKs signal pathway but not PI-3K, p38 MAPK and JNKs pathways in PMA-treated HL-60 cells. HL-60 cells were treated with PKC (5 µM), ERKs (5 µM), PI-3K (200 nM), p38 MAPK (10 µM), and JNKs (20 µM) inhibitors in the absence or presence of PMA (30 nM) for 24 h in serum free RPMI-1640 medium, respectively. Total RNA from these cells was isolated and GM3 synthase mRNA was detected by RT-PCR and northern blot analysis. Beta-actin was included as an internal control (A). Protein lysates from these cells were prepared. Phosphorylation of the indicated proteins were determined by western blot analysis using antibodies recognizing only phosphorylated forms of ERK1/2, AKT, p38 MAPK, and JNKs. GAPDH was included as an internal control (B).

Fig. 2.

The enhanced expression of GM3 synthase through PKC/ERKs signal pathway but not PI-3K, p38 MAPK and JNKs pathways in PMA-treated HL-60 cells. HL-60 cells were treated with PKC (5 µM), ERKs (5 µM), PI-3K (200 nM), p38 MAPK (10 µM), and JNKs (20 µM) inhibitors in the absence or presence of PMA (30 nM) for 24 h in serum free RPMI-1640 medium, respectively. Total RNA from these cells was isolated and GM3 synthase mRNA was detected by RT-PCR and northern blot analysis. Beta-actin was included as an internal control (A). Protein lysates from these cells were prepared. Phosphorylation of the indicated proteins were determined by western blot analysis using antibodies recognizing only phosphorylated forms of ERK1/2, AKT, p38 MAPK, and JNKs. GAPDH was included as an internal control (B).

To further determine which signal transduction pathway is involved in PMA-stimulated induction of GM3 sythase in HL-60 differentiation, we investigated the activities of signal pathways that were assessed by measuring their degree of regulatory phosphorylation using phospho-specific antibodies. As shown in Figure 2B, to determine whether transcriptional activation of GM3 synthase through PKC/ERKs pathway in HL-60 cells induced by PMA, we performed western blot analysis. Phosphorylation of ERK1/2, a downstream activator of PKC, was significantly elevated in PMA-stimulated HL-60 cells than in uninduced HL-60 cells. However, PKC inhibitor GÖ6976 and ERK inhibitor U0126 specially blocked PMA-stimulated activation of ERK1/2, respectively.

In our previous data, GÖ6976 and U0126 inhibitors have been also shown to block PMA-induced expression of GM3 synthase in HL-60 cells. On the other hand, AKT, which is known as a downstream activator of PI-3K and p38 MAPK were not activated by PMA. Treatment of HL-60 cells with PI-3K inhibitor wortmannin or p38 MAPK inhibitor SB203580 in presence and absence of PMA reduced AKT and p38 MAPK phosphorylation, respectively, compared with PMA-induced or uninduced HL-60 cells. Both wortmannin and SB203580 did not affect PMA-stimulated GM3 synthase expression. Furthermore, although JNK inhibitor SP600125 decreased phosphorylation of JNKs in PMA-activated HL-60 cells, PMA-induced expression of GM3 synthase was not inhibited by SP600125. These results suggest that PMA-induced HL-60 cells modulate transcriptional activation of GM3 synthase through PKC/ERKs pathway but not PI-3K, p38 MAPK and JNKs pathways.

Transcriptional activation of GM3 synthase through PKC/ERKs-dependent CREB activation in PMA-induced HL-60 cells

To determine the region regulating the transcription activity of the GM3 synthase during PMA-induced differentiation of HL-60 cells, a genomic fragment containing 1600 bp of 5′-promoter region of GM3 synthase gene was subcloned into pGL3-basic vector (pGL3-1600). The luciferase constructs carrying 5′-deleted GM3 synthase promoter (pGL3-1210, pGL3-847, pGL3-432, pGL3-177, and pGL3-83) were also prepared. After transfecting the constructed plasmids into HL-60 cells, we checked GM3 synthase promoter activity induced by PMA. As shown in Figure 3A, deletion of nucleotide −1600 to −83 had no significant effect on transcriptional activity in PMA-untreated HL-60 cells as measured by luciferase promoter assay. However, in PMA-induced cells, transcriptional activities were significantly higher than in untreated control cells and gradual deletion of 5′-sequences from nucleotides −1600 to −177 resulted in about fourfold increase in transcriptional activity compared with the promoterless and enhancerless control vector pGL3-basic. The maximum activity was obtained with the pGL3-177 and reached about eightfold higher activity than the pGL3-basic. Further deletion to nucleotide −83 markedly reduced transcriptional activity to the similar level of the control vector pGL3-basic. These results clearly show that the region from nucleotides −177 to −83 functions as the PMA-inducible promoter in HL-60 cells.

Fig. 3.

Transcriptional activation of GM3 synthase through PKC/ERKs-dependent CREB activation in PMA-induced HL-60 cells. Schematic representation of DNA constructions containing various lengths, and CREB binding site mutants of the 5′-flanking region of GM3 synthase linked to the luciferase reporter gene are presented. The restriction sites are shown, and the transcription start site is indicated as +1. The pGL3-basic without any promoter and enhancer was used as negative control. The pGL3-control with SV40 promoter and enhancer was used as positive control. Each construct was cotransfected into HL-60 cells with pCMVβ as the internal control. The transfected cells were incubated in the presence (solid bar) and absence (open bar) of 30 nM TPA for 24 h. Relative luciferase activity was normalized with β-galactosidase activity derived from pCMVβ. The values represent the mean ± SD for three independent experiments with triplicate measurements (A). The pGL3-177 was cotransfected into HL-60 cells with pCMVβ as the internal control. The transfected cells were incubated in the presence (solid bar) and absence (open bar) of 30 nM TPA with PKC (5 µM), ERKs (5 µM), PI-3K (200 nM), p38 MAPK (10 µM) and JNK (20 µM) inhibitors for 24 h. Relative luciferase activity was normalized with β-galactosidase activity derived from pCMVβ. The values represent the mean ± SD for three independent experiments with triplicate measurements (B). HL-60 cells were treated with PMA (30 nM) in the presence of PKC (5 µM), ERKs (5 µM), PI-3K (200 nM), p38 MAPK (10 µM), and JNK (20 µM) inhibitors for 24 h in serum free RPMI-1640 medium, respectively. Protein lysates from these cells were prepared. Phosphorylation of the indicated proteins were determined by western blot analysis using antibodies recognizing only phosphorylated forms of CREB, c-Jun, ATF-2. GAPDH was included as an internal control (C). HL-60 cells were treated with PMA in the presence of chemical inhibitors for 24 h in serum-free RPMI-1640 medium, respectively. Nuclear extracts isolated from these cells were incubated with 32P-labeled wild-type probe. For gel supershift analysis, nuclear extracts were incubated with 32P-labeled wild-type probe in the presence of anti-CREB, anti-c-Jun, and anti-ATF-2 antibodies, respectively. For competition experiments, 50-fold molar excess of unlabeled wild-type or unlabeled mutant CREB oligonucleotides was used. The DNA–protein complexes were analyzed on a 4% nondenaturing polyacrylamide gel (D).

Fig. 3.

Transcriptional activation of GM3 synthase through PKC/ERKs-dependent CREB activation in PMA-induced HL-60 cells. Schematic representation of DNA constructions containing various lengths, and CREB binding site mutants of the 5′-flanking region of GM3 synthase linked to the luciferase reporter gene are presented. The restriction sites are shown, and the transcription start site is indicated as +1. The pGL3-basic without any promoter and enhancer was used as negative control. The pGL3-control with SV40 promoter and enhancer was used as positive control. Each construct was cotransfected into HL-60 cells with pCMVβ as the internal control. The transfected cells were incubated in the presence (solid bar) and absence (open bar) of 30 nM TPA for 24 h. Relative luciferase activity was normalized with β-galactosidase activity derived from pCMVβ. The values represent the mean ± SD for three independent experiments with triplicate measurements (A). The pGL3-177 was cotransfected into HL-60 cells with pCMVβ as the internal control. The transfected cells were incubated in the presence (solid bar) and absence (open bar) of 30 nM TPA with PKC (5 µM), ERKs (5 µM), PI-3K (200 nM), p38 MAPK (10 µM) and JNK (20 µM) inhibitors for 24 h. Relative luciferase activity was normalized with β-galactosidase activity derived from pCMVβ. The values represent the mean ± SD for three independent experiments with triplicate measurements (B). HL-60 cells were treated with PMA (30 nM) in the presence of PKC (5 µM), ERKs (5 µM), PI-3K (200 nM), p38 MAPK (10 µM), and JNK (20 µM) inhibitors for 24 h in serum free RPMI-1640 medium, respectively. Protein lysates from these cells were prepared. Phosphorylation of the indicated proteins were determined by western blot analysis using antibodies recognizing only phosphorylated forms of CREB, c-Jun, ATF-2. GAPDH was included as an internal control (C). HL-60 cells were treated with PMA in the presence of chemical inhibitors for 24 h in serum-free RPMI-1640 medium, respectively. Nuclear extracts isolated from these cells were incubated with 32P-labeled wild-type probe. For gel supershift analysis, nuclear extracts were incubated with 32P-labeled wild-type probe in the presence of anti-CREB, anti-c-Jun, and anti-ATF-2 antibodies, respectively. For competition experiments, 50-fold molar excess of unlabeled wild-type or unlabeled mutant CREB oligonucleotides was used. The DNA–protein complexes were analyzed on a 4% nondenaturing polyacrylamide gel (D).

To confirm whether this site contributes to PMA-induced expression of GM3 synthase in HL-60 cell, pGL3-1600mtCREB and pGL3-177mtCREB were constructed by mutating CREB site from pGL3-1600 and pGL3-177, respectively. In PMA-treated HL-60 cells, these mutations markedly reduced transcriptional activity to the similar level of the promoterless and enhancerless control vector pGL3-Basic (Figure 3A). These results indicate that the expression of GM3 synthase is mediated through CREB binding site of GM3 synthase promoter in PMA-stimulated HL-60 cells.

The previous results showed that expression of GM3 synthase was associated with PKC/ERKs pathway in PMA-induced HL-60 cells. Thus we also investigated whether PMA-induced transcriptional activity of a pGL3-177-containing CREB binding site was stimulated via PKC/ERKs signal pathway. As shown in Figure 3B that activity of pGL3-177 was increased in PMA-stimulated HL-60 cells, compared with untreated HL-60 cells. However, both PKC and ERKs inhibitors markedly resulted in a decrease of pGL3-177 activity in HL-60 cells induced by PMA. On the other hand, activity of pGL-177 in PMA-stimulated HL-60 cells was not significantly inhibited by PI-3K, p38 MAPK, and JNK inhibitors, respectively, compared with HL-60 cells induced by PMA in absence of chemical inhibitors, as evidenced by luciferase promoter assay. These results show that promoter activity as well as mRNA level of GM3 synthase was regulated by PKC/ERKs signaling pathway.

To further confirm that activation of PKC/ERKs pathway in PMA-stimulated HL-60 cells is directly involved in the CREB-mediated transcriptional activation of GM3 synthase, we examined whether CREB, as a transcriptional activator, is activated in PMA-induced HL-60 through PKC/ERKs pathway using western blot analysis. We also examined whether activation of PKC/ERKs pathway during the differentiation of HL-60 cells stimulated by PMA is correlated to the binding of PMA-activated CREB to wild-type oligoncleotides that contain the sequence for the CREB binding site from the promoter of GM3 synthase using EMSA.

As shown in Figure 3C, PMA-stimulated HL-60 cells increased CREB phosphorylation compared with PMA-uninduced HL-60 cells. However, PKC and ERK inhibitors blocked PMA-stimulated CREB phosphorylation, whereas activation of CREB in PMA-stimulated HL-60 cells was not prevented by PI-3K, p38 MAPK, and JNKs inhibitors, respectively, compared with HL-60 cells induced by PMA in absence of chemical inhibitors, as evidenced by western blot analysis. In addition, as shown in Figure 3D, the intensity levels of the CREB-shifted bands in the nuclear lysates from PMA-induced HL-60 cells were higher than those for the nuclear lysates from PMA-uninduced HL-60 cells, as evidenced by EMSA. Moreover, the formation of an electrophoretically retarded complex was inhibited only when an unlabeled wild-type oligonucleotide (wt oligo) was introduced, but not when an oligonucleotide with mutations in each of the half-sites (mut oligo) was introduced. However, PKC and ERK inhibitors significantly blocked the formation of an electrophoretically retarded complex in PMA-stimulated HL-60 cells. PMA-induced electromobility shift in HL-60 cells was not decreased by PI-3K, p38 MAPK, and JNK inhibitors, respectively, compared with HL-60 cells induced by PMA in absence of chemical inhibitors. These results clearly show that transcriptional activation of GM3 synthase was induced through PKC/ERKs-dependent CREB activation in PMA-induced HL-60 cells.

We further investigated whether p38 MAPK or JNKs phosphorylate CREB, c-Jun, or ATF-2 in HL-60 cells stimulated by PMA, and transcriptional activity of GM3 synthase was affected by activated CREB, c-Jun, or ATF-2. As shown in Figure 3C, although p38 MAPK inhibitor blocked p38 MAPK activation in PMA-stimulated HL-60 cells, activation of CREB, c-Jun, and ATF-2 was not affected by p38 MAPK inhibitor. On the other hand, JNKs activation in PMA-stimulated HL-60 cells induced an increase of c-Jun phosphorylation except for CREB and ATF-2 activation. To clarify whether the shift band of DNA–protein complex contained CREB activated by PKC/ERKs or c-Jun activated by JNKs, we also performed EMSA in the presence of anti-CREB, anti-c-Jun, or anti-ATF-2 antibodies. The incubation of nuclear extracts with anti-CREB antibody resulted in a supershift of the complex with a concomitant diminution of the retarded band, both anti-c-Jun and anti-ATF-2 antibody (Figure 3D). We first demonstrate that PKC/ERKs-dependent CREB activation is necessary for expression of GM3 synthase in HL-60 cells induced by PMA.

Effect of increased ganglioside GM3 through PKC/ERKs-dependent CREB activation and exogenous GM3 on CD11b expression and cell adherence

We first investigated effect of ganglioside GM3 on expression of CD11b as a differentiation marker of HL-60 cells. Our previous data have clearly showed that transcriptional activity of GM3 synthase was induced through PKC/ERKs-dependent CREB activation in PMA-stimulated HL-60 cells. As shown in Figure 4A, GM3 was produced through PKC/ERKs pathway as well. Furthermore, the pattern of CD11b expression depended on increased expression of GM3 synthase and ganglioside GM3 by PKC/ERKs activation (Figure 4B). Moreover, to further clarify whether endogenous and exogenous ganglioside GM3 affect expression of CD 11b, which is known as monocyte/macrophage differentiation marker, we checked CD11b expression levels after treatment of HL-60 cells with L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), a glucosylceramide synthase inhibitor, in the presence of PMA and exogenous GM3.

Fig. 4.

Effect of the increased ganglioside GM3 through PKC/ERKs-dependent CREB activation on expression of CD11b surface antigen. HL-60 cells were treated with PKC (5 µM), ERKs (5 µM), PI-3K (200 nM), p38 MAPK (10 µM), and JNK (20 µM) inhibitors in the absence or presence of PMA (30 nM) for 24 h. Glycolipid isolated from these cells was applied and developed on a HPTLC plate with chloroform-methanol-water (60:40:9). After separation of gangliosides on HPTLC, gangioside GM3 was detected by HPTLC immunostaining with M2590 antibody, as shown in Materials and methods (A). Total RNA from these cells was isolated and CD11b expression was detected by RT-PCR. Beta-actin was included as an internal control (B).

Fig. 4.

Effect of the increased ganglioside GM3 through PKC/ERKs-dependent CREB activation on expression of CD11b surface antigen. HL-60 cells were treated with PKC (5 µM), ERKs (5 µM), PI-3K (200 nM), p38 MAPK (10 µM), and JNK (20 µM) inhibitors in the absence or presence of PMA (30 nM) for 24 h. Glycolipid isolated from these cells was applied and developed on a HPTLC plate with chloroform-methanol-water (60:40:9). After separation of gangliosides on HPTLC, gangioside GM3 was detected by HPTLC immunostaining with M2590 antibody, as shown in Materials and methods (A). Total RNA from these cells was isolated and CD11b expression was detected by RT-PCR. Beta-actin was included as an internal control (B).

As shown in Figure 5A, both GM3 synthase and CD11b expression were induced in PMA-stimulated HL-60 cells. However, because PDMP resulted in a decrease of ganglioside GM3 by removing GM3 precusor in PMA-induced HL-60 cells, CD11b expression was decreased although PDMP had no effect on PMA-induced expression of GM3 synthase in HL-60 cells. The suspension cells exhibit morphological characteristics of differentiated monocytes, inducing adherence to the tissue culture plate and irregular cellular shape during the differentiation of HL-60 cells induced by PMA. As shown in Figure 5A, the HL-60 cells differentiated by PMA resulted in the increase of cellular adhesion and altered morphology, which is characteristic of monocytic differentiation. However, PDMP blocked cellular adhesion of HL-60 cells. In addition, treatment of HL-60 cells with ganglioside GM3 (30 mM) affected CD11b expression (Figure 5B) and adherence to the tissue culture plate (data not shown). These results indicate that endogenous and exogenous ganglioside GM3 modulate expression of CD11b known as monocyte/macrophage differentiation maker and induce the attachment of suspension cells to the tissue culture plate.

Fig. 5.

Effect of endogenous and exogenous ganglioside GM3 on CD11b expression and cell adherence. HL-60 cells were cultured in serum-free RPMI 1640 medium containing PMA (30 nM) and/or PDMP (10 µM). Total RNA from these cells was isolated and GM3 synthase and CD11b mRNA was detected by RT-PCR. Beta-actin was included as an internal control. In addition, glycolipids from HL-60 cells were isolated after 24 h of PMA and/or PDMP treatment. After separation of gangliosides on HPTLC, gangioside GM3 was detected by HPTLC immunostaining with M2590 antibody (A). HL-60 cells were cultured in serum-free RPMI 1640 medium containing PMA and/or PDMP. The medium was removed, and any remaining nonadherent cells were gently washed away with PBS and the adherent cells fixed with 10% paraformaldehyde for 1 h. The wells were washed twice with PBS and stained with 5% Giemsa in PBS overnight. The wells were then washed with PBS and air-dried. Photographs were taken with a microscope (B). HL-60 cells were treated with PMA or ganglioside GM3 (30 µM). Total RNA from these cells was isolated and CD11b mRNA was detected by RT-PCR. Beta-actin was included as an internal control (C).

Fig. 5.

Effect of endogenous and exogenous ganglioside GM3 on CD11b expression and cell adherence. HL-60 cells were cultured in serum-free RPMI 1640 medium containing PMA (30 nM) and/or PDMP (10 µM). Total RNA from these cells was isolated and GM3 synthase and CD11b mRNA was detected by RT-PCR. Beta-actin was included as an internal control. In addition, glycolipids from HL-60 cells were isolated after 24 h of PMA and/or PDMP treatment. After separation of gangliosides on HPTLC, gangioside GM3 was detected by HPTLC immunostaining with M2590 antibody (A). HL-60 cells were cultured in serum-free RPMI 1640 medium containing PMA and/or PDMP. The medium was removed, and any remaining nonadherent cells were gently washed away with PBS and the adherent cells fixed with 10% paraformaldehyde for 1 h. The wells were washed twice with PBS and stained with 5% Giemsa in PBS overnight. The wells were then washed with PBS and air-dried. Photographs were taken with a microscope (B). HL-60 cells were treated with PMA or ganglioside GM3 (30 µM). Total RNA from these cells was isolated and CD11b mRNA was detected by RT-PCR. Beta-actin was included as an internal control (C).

Discussion

Previous studies have shown that activity of GM3 synthase, inducing an increase in ganglioside GM3 content, was significantly increased during the differentiation of PMA-induced HL-60 cells (Momoi and Yokota, 1983; Momoi et al., 1986; Nojiri et al., 1984). However, transcriptional regulation of GM3 synthase during PMA-induced differentiation of HL-60 cells had not been examined at the molecular level. We report for the first time that the expression of GM3 synthase is up-regulated during the differentiation of PMA-induced HL-60 cells. Treatment of HL-60 cells with PMA resulted in an increase of GM3 synthase mRNA levels in a time-dependent manner. Ganglioside GM3 is synthesized by GM3 synthase, which catalyzes the transfer of NeuAc of CMP-NeuAc to the nonreducing terminal galactose of lactosylceramide.

Several studies have shown that the content of ganglioside GM3 was increased in PMA-stimulated HL-60 cells (Momoi et al., 1986; Xia et al., 1989). Our previous data showed that PMA-induced HL-60 cells result in the increase of GM3 synthase expression at mRNA levels. We also observed the increased levels of ganglioside GM3 in a time-dependent manner during the differentiation of PMA-induced HL-60 cells. Recently, we isolated and functionally characterized the GM3 synthase promoter in human hepatoma HepG2 and neuroblastoma SK-N-MC cells (Kim et al., 2002). Thus, we first investigated if the promoter activity of GM3 synthase was stimulated during HL-60 differentiation by PMA treatment. the promoter activity of GM3 synthase in PMA-stimulated HL-60 cells was about 4.5-fold higher than that in PMA-uninduced HL-60 cells. Therefore, our present data clearly show that both transcriptional activity of GM3 synthase and its product GM3 are increased in PMA-stimulated HL-60 cells.

Several studies have reported changes in the expression of the PKC-α and PKC-β gene during the chemically induced differentiation of HL-60 cells (Aihara et al., 1991; Macfarlane and Manzel 1994; McSwine-Kennick et al., 1991; Nishikawa et al., 1990). Tonetti et al. (1994) have also reported that PKC-β is one of the essential elements in the PMA-induced signal transduction pathway, which leads to macrophage differentiation in HL-60 cells using the variant HL-525 cells deficient in PKC-β and resistant to PMA-induced differentiation. Our data also show that the phosphorylation of PKC/ERKs as well as JNKs was induced in PMA-differentiated HL-60 cells, as measured by western blot analysis.

Although our previous data demonstrated that PKC/ERKs activation and GM3 expression were increased during the differentiation of PMA-induced HL-60 cells, the transcriptional activation of GM3 synthase directly associated with PKC/ERKs pathway during HL-60 cells differentiation induced by PMA, which has not been reported yet. In this study, we confirmed that GM3 synthase expression and ERKs phosphorylation are induced in the differentiated HL-60 cells by PMA. However, PKC and ERK inhibitors blocked GM3 sythase expression as well as ERK activation. On the other hand, PI-3K, p38 MAPK, and JNK inhibitors did not decrease the expression levels of GM3 synthase stimulated by PMA during the differentiation of HL-60 cells. These results suggest that transcriptional activity of GM3 synthase may be related to PKC/ERKs-dependent pathway.

We also conclude that the region between −177 and −83 in the GM3 synthase promoter functions as the core promoter essential for transcriptional activation of GM3 synthase in PMA-induced HL-60 cells. We have previously reported that this region was also needed for the enhancer activity of the GM3 synthase promoter in SK-N-MC and HepG2 cells (Kim et al., 2002). Endogenous GM3 synthase gene expression was detected in both cell lines by RT-PCR (Kim et al., 2002) but not in PMA-uninduced HL-60 cells. This indicates that this region functions as the PMA-inducible promoter during the differentiation of HL-60 cells. Our previous study also revealed the existence of several transcription factor binding sites such as NFY, CREB, SP1, EGR3, and MZF1 in this region (Kim et al., 2002). Recent reports have suggested that only the consensus CREB binding site (TGACGTCA) at position −143 to −136 in this region might contribute to GM3 synthase promoter activity (Choi et al., 2003, 2004; Zeng et al., 2003). Our present findings, by site-directed mutagenesis, indicate that this CREB element mediates PMA-dependent up-regulation of GM3 synthase expression, as evidenced by luciferase promoter assay.

CREB, a transcription factor, is the target of a variety of signaling pathways mediating cell responses to extracellular stimuli, involving proliferation, differentiation, and adaptive responses of cell processes (De Cesare et al., 1999; Shaywitz and Greenberg 1999). Several signal pathways, such as PKA, PKC, Ca2+/CaMKs, SAPK/JNK, p38 MAPK, and ERKs could activate CREB binding to CRE, and the capacity of CREB to activate transcription is regulated by phosphorylation at serine 133 (De Cesare et al., 1999; Shaywitz and Greenberg 1999). Previous work has shown that the activation of PKC by PMA in human fibroblasts stimulates CREB phosphorylation and subsequent CREB-mediated gene transcription (Manier et al., 2001). Other studies have indicated that CREB phosphorylation in oligodendrocytes may be mediated by PKC and MAPK-dependent signal transduction pathways (McNulty et al., 2001; Pende et al., 1997; Sato-Bigbee et al., 1999). Therefore, to further clarify that activation of the PKC/ERKs pathway in PMA-stimulated HL-60 cells is directly involved in the CREB-mediated transcriptional activation of GM3 synthase, we performed western blot analysis to measure regulatory phosphorylation using chemical inhibitors of CREB upstream signal activator.

EMSA was performed using oligonucleotides that contain the sequence for the CREB binding site from the GM3 synthase promoter, and luciferase promoter assay used a pGL3-177 containing CREB binding site from GM3 synthase promoter. Thus our results clearly demonstrate that CREB phosphorylation in PMA-stimulated HL-60 cells is mediated by PKC/ERKs-dependent pathway but not PI-3K, p38 MAPK, and JNKs pathways. Furthermore, we have demonstrated that the activated CREB binds to the site of the GM3 synthase promoter through PKC/ERKs-dependent pathway in PMA-induced HL-60 cells. Moreover, the activity of pGL3-177, containing CREB binding site at position −143 to −136 of 5′-flanking region of GM3 synthase promoter was regulated by PKC/ERKs-dependent pathway in PMA-induced HL-60 cells. As such, we demonstrate that the activation of PKC/ERKs signal pathway modulates CREB-mediated transcriptional activation of GM3 synthase during the differentiation of PMA-stimulated HL-60 cells.

Kim et al. (2002) reported that the consensus ATF/CREB site, which is recognized by a family of proteins referred to as ATF or CREB, is completely conserved at position −143 to −136 in promoter region of GM3 synthase. Furthermore, analysis of the 5′-flanking region of GM3 synthase gene by the MatInspector v2.2 program (core similarity 1.0, matrix similarity 0.95) using TRANSFAC 4.0 matrices revealed the presence of CREBP1CJUN, which is recognized by CRE-binding protein and c-Jun heterodimer at the same position with CREB binding site. It is possible, therefore, that the expression of GM3 synthase gene may be activated by PKC and MAPKs signaling pathway. Several studies showed that the RAP1/PKC/ERKs-dependent pathway induces the phosphorylation of ATF-1, CREB, and Elk-1, whereas the p38 MAPK-dependent pathway only causes CREB phosphorylation (Schinelli et al., 2001). In addition, JNKs phosphorylate the NH2-terminal activation domain of c-Jun and ATF-2, increasing their transcriptional activity (Derijard et al., 1994; Kyriakis and Avruch 1996). Moreover, p38 MAPK activates c-Jun and CREB (Tan et al., 1996; Zhang et al., 1999). Macrophage differentiation of HL-60 cells induced by PMA resulted in marked increase in c-Jun mRNA (Gaynor et al., 1991). Activated c-Jun has been described in several studies to heterodimerize with CREB (Benbrook and Jones 1990; Chevray and Nathans 1992; Hai and Curran 1991). These results suggest that one of these complexes may contain c-Jun heterodimer with CREB. Our previous result also shows that c-Jun is activated by JNKs in PMA-stimulated HL-60 cells. In addition, we found that CREB activation was increased by stimulation of PKC/ERKs pathway. However, phosphorylation of ATF was not induced in PMA-stimulated HL-60 cells. Therefore, to clarify which complex contains CREB, c-Jun, ATF, c-Jun heterodimer with CREB, and ATF heterodimer with CREB, we further performed gel supershift assay using anti-CREB, anti-c-Jun, and anti-ATF antibodies. Our data clearly show that the incubation of nuclear extracts with anti-CREB antibody results in a supershift of DNA–protein complex with a concomitant diminution of the retarded band, but not anti-ATF and c-Jun antibody. Thus, PKC/ERKs-dependent CREB activation is a prerequisite essential to the transcriptional activation of GM3 synthase in PMA-induced HL-60 cells.

Expression of CD11b subunit depends on the stage of differentiation with mature macrophages expressing the highest levels of CD11b surface antigen and mRNA. It has been shown that expression of CD11b was increased in PMA-differentiated HL-60 cells (Aihara et al., 1991). Our present data reveal that ganglioside GM3 as well as transcriptional activation of GM3 synthase through the PKC/ERKs-dependent pathway was increased in the PMA-diffrentiated HL-60 cells. Additionally, ganglioside GM3 induces the maturation of HL-60 cells along monocytic/macrophage lineage (Nojiri et al., 1986; Stevens et al., 1989). However, CD11b expression by PMA-induced ganglioside GM3 and exogenous GM3 in HL-60 cells is not well understood. Interestingly, our present results demonstrate that ganglioside GM3 induced by activation of PKC/ERKs pathway is related to expression of CD11b, a differentiation marker. Furthermore, the depletion of ganglioside GM3 by treating PMA-induced cells with PDMP, a glucosylceramide synthase inhibitor, resulted in a significant reduction of CD11b expression as well as cellular adhesion. Moreover, ganglioside GM3-treated HL-60 cells induced expression of the CD11b differentiation marker.

In conclusion, as illustrated by Figure 6, PMA induces the marked activation of the PKC/ERKs signal transduction pathway during the differentiation of HL-60 cells. PKC/ERKs activation leads to the phosphorylation of CREB as a transcription factor. In PMA-stimulated HL-60 cells, the PKC/ERKs-dependent CREB activation regulates expression of GM3 synthase, inducing a synthesis of the ganglioside GM3 product. Furthermore, the increased expression of GM3 through PKC/ERKs/CREB-dependent pathway and exogenous GM3 result in an increase of CD11b surface antigen expression. Based on these results, we have demonstrated that the ganglioside GM3, which is induced through PKC/ERKs/CREB-dependent pathway, takes part in the differentiation of HL-60 cells by inducing expression of CD 11b, known as a monocyte/macrophage differentiation maker.

Fig. 6.

The molecular mechanisms involved in transcriptional activation of GM3 synthase during the differentiation of PMA-induced HL-60 cells and the function of ganglioside GM3 in the differentiation of HL-60 cells. PMA induce the marked activation of the PKC/ERKs signal transduction pathway during the differentiation of HL-60 cells. PKC/ERKs activation induced by PMA, in HL-60 cells, leads to the phosphorylation of CREB as a transcription factor. In PMA-stimulated HL-60 cells, the PKC/ERKs-dependent CREB activation regulates expression of GM3 synthase, inducing a synthesis of ganglioside GM3 product. Furthermore, the increased ganglioside GM3 through PKC/ERKs/CREB-dependent pathway result in an increase of CD11b surface antigen expression.

Fig. 6.

The molecular mechanisms involved in transcriptional activation of GM3 synthase during the differentiation of PMA-induced HL-60 cells and the function of ganglioside GM3 in the differentiation of HL-60 cells. PMA induce the marked activation of the PKC/ERKs signal transduction pathway during the differentiation of HL-60 cells. PKC/ERKs activation induced by PMA, in HL-60 cells, leads to the phosphorylation of CREB as a transcription factor. In PMA-stimulated HL-60 cells, the PKC/ERKs-dependent CREB activation regulates expression of GM3 synthase, inducing a synthesis of ganglioside GM3 product. Furthermore, the increased ganglioside GM3 through PKC/ERKs/CREB-dependent pathway result in an increase of CD11b surface antigen expression.

Materials and methods

Cell line and cell culture

The HL-60 cell line was obtained form American Type Culture Collection (Rockville, MD). This cell line was maintained in RPMI 1640 medium (Gibco, Rockville, MD) supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C under 5% CO2.

RT-PCR and northern blot analysis

Total RNA was isolated from HL-60 cells treated with or without PMA in presence of signal inhibitors using Trizol reagent (Invitrogen, Carlsbad, CA). Two micrograms of RNA was subjected to reverse transcription with random nonamers using Takara RNA PCR kit (Takara, Japan) according to the manufacturer's protocol. The cDNA was amplified by PCR with the following primers: GM3 synthase (413 bp), 5′-CCCTGCCATTCTGGGTACGAC-3′ (sense) and 5′-CACGATCAATGCCTCCACTGAGATC-3′ (antisense); CD11b (570 bp), 5′-ATGGCTCTCAGAGTCCTTCTGTTAA-3′ (sense) and 5′-CATCAAAGAGAACAAGGTTTTGGAC-3′ (antisense); β-actin (247 bp), 5′-CAAGAGATGGCCACGGCTGCT-3′ (sense) and 5′-TCCTTCTGCATCCTGTCGGCA-3′ (antisense). The PCR products were separated by gel electrophoresis on 3% agarose containing ethidium bromide with 1× TAE buffer. To assess the specificity of the amplification, the PCR product for GM3 synthase was subcloned into pGEM-T vector (Promega, Madison, WI) and then sequenced. These genes were found to be identical to the expected cDNA. Northern blot analysis was performed by the same method as described previously (Chung et al., 2003), using the [α-32P]dCTP-labeled fragments of GM3 synthase as a probe.

HPTLC immunostaining

Glycolipids were applied and developed on HPTLC plates with chloroform-methanol-water (60:40:9). After separation of gangliosides, HPTLC plates were coated with an n-hexane solution containing 0.1% polyisobutylmethacrylate for 1 min and completely dried. The dried plates were incubated in phosphate buffered saline (PBS) containing 1% bovine serum albumin (1% BSA/PBS) for 1 h at room temperature, and then the plates were incubated in 1% BSA/PBS containing M2590 antibody (Biotest Laboratories, Japan) at RT for 2 h. The plates were washed with PBS containing 0.05% Tween 20 (PBS-T) three times, and incubated in a horseradish peroxidase–conjugated goat anti-mouse IgM (Biomeda, Foster City, CA) solution in 1% BSA/PBS for 1 h at room temperature. After washing the plates with PBS-T, a solution of Chemiluminescence Reagent Kit (Amersham Biosciences, Little Chalfont, U.K.) was added to the plate, followed by exposure to X-ray film for 30 s and visualization of the spot by development.

Western blot analysis

Cells were homogenized in a sample buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 3 mM NaN3, 0.57 mM phenylmethylsulfonyl fluoride, 0.15 µM aprotinin, and 1% Triton X-100. Protein concentrations were measured using the Bio-Rad protein assay (Bio-Rad, Hercules, CA). Twenty-microgram samples of total cell lysates were size fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose membranes using the Hoefer electrotransfer system (Amersham Biosciences). To detect p-ERK, ERK, p-AKT, p-CREB, CREB, p-p38, p-JNK, p-ATF, p-c-Jun, and GAPDH protein, the membranes were incubated with p-ERK (Cell Signal, Beverly, MA), ERK (Cell Signal), p-AKT (Santa Cruz, Santa Cruz, CA), p-CREB (Upstate, Lake Placid, NY), CREB (Upstate), p-p38 (Cell Signal), p-JNK (Cell Signal), p-ATF (SantaCruz), p-c-Jun (SantaCruz), and GAPDH antibodies (Chemicon, Temecula, CA). Detection was performed using a secondary horseradish peroxidase–linked anti-mouse antibody and the chemiluminescence system (Amersharm Biosciences).

Cellular adherence assay

HL-60 cells were incubated with various chemical inhibitors in presence or absence of PMA for 24 h. The medium was removed, and any remaining nonadherent cells were gently washed away with PBS and the adherent cells fixed with 10% paraformaldehyde for 1 h. The wells were washed twice with PBS and stained with 5% Giemsa in PBS overnight. The wells were then washed with PBS and air-dried. Photographs were taken with a light microscope at 20× magnification.

Preparation of reporter plasmids and mutagenesis

Reporter plasmids, pGL3-1600 and its derivatives (pGL3-83 to pGL3-1210) were prepared by insertion of the SacI/BglII fragments of the each plasmid constructed previously (Kim et al., 2002) into the corresponding sites of the promoter-less luciferase vector pGL3-Basic (Promega). Mutation with base substitution at CREB binding site was accomplished using a QuikChange II XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's protocol with the following oligonucleotide primers: CREB-L, 5′-GTCCTCGTGTTGTCAGACCCCGCCCACGCGCCCCT-3′ and CREB-R, 5′-CGGGGTCTGACAACACGAGGACGCGGACGGCCAAT-3′ (mutated nucleotides underscored). The presence of mutation was verified by sequence analysis.

Transfection and luciferase assay

For the reporter analysis of GM3 synthase promoter, transient transfection of HL-60 cells was carried out by electroporation. Briefly, the cultured cells were washed with PBS buffer and were then centrifuged at 150 × g for 5 min at room temperature. The cells were washed in Puck's saline buffer containing 137 mM NaCl, 5.4 mM MgCl2, 4.2 mM NaHCO3, and 5.5 mM glucose before being suspended in permeabilization buffer (20 mM HEPES pH 7.05, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 6 mM dextrose) containing 10 µg of the luciferase reporter constructs and 10 µg of a cytomegalovirus-β-galactosidase vector (pCMVβ) as a transfection efficiency control. Cells (2.5 × 106) were suspended in permeabilization buffer and placed in a cuvette. Electroporation was accomplished using a Bio-Rad Gene Pulser II at 500 μF and 300 V. To stimulate resealing of the cell after electrophoration, the cells were incubated for 1 h at 37°C in 5% CO2. The cells were then resuspended in RPMI 1640 medium containing 10% fetal bovine serum and cultured for 12 h. PMA (30 nM) was then added to induce cell differentiation, and cells were cultured for another 24 h. Cells were then harvested, and luciferase activity was measured using the dual-luciferase reporter assay system kit (Promega) and Luminoskan Ascent (Thermo Labsystems, Finland). Luciferase activity was normalized to β-galactosidase activity.

EMSA

Nuclear extracts from resting and PMA-induced HL-60 cell were prepared as described previously (Chung et al., 2003). EMSAs were performed using gel shift assay system kit (Promega) according to the manufacturer's instructions. Briefly, double-stranded oligonucleotides containing the consensus sequence for CREB (5′-TCCTCGTGACGTCAGACCCC-3′) were end-labeled with [γ-32P]ATP using T4 polynucleotide kinase and used as probes for EMSA. Competition was performed using the unlabeled wild-type CREB or a mutant oligomer 5′-TCCTCGTGTTGTCAGACCCC-3′ (mut CREB). Nuclear extract proteins (2 µg) were preincubated with the gel shift binding buffer [4% glycerol, 1 mM MgCl2, 0.5 mM ethylenediamine tetra-acetic acid, 0.5 mM dithiothreitol, 50 mM NaCl, 10 mM Tris–HCl (pH 7.5), and 0.05 mg/ml poly (deoxyinosine-deoxycytosine)] for 10 min and then incubated with the labeled probe for 20 min at room temperature. Each sample was electrophoresed in a 4% nondenaturing polyacrylamide gel in 0.5× TBE buffer at 250 V for 20 min. The resulting gel was dried and subjected to autoradiography.

This work was supported by National Research Laboratory Program (M10203000024-02J0000-01300) from the Ministry of Science and Technology, Korea (C.H.K).

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Author notes

2National Research Laboratory for Glycobiology, MOST, Korea and Department of Biochemistry and Molecular Biology, College of Oriental Medicine, Dongguk University, Kyungju 780-714, South Korea; 3Faculty of Biotechnolgy, Dong-A University, Pusan 604–714, South Korea