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Cell Growth & Differentiation Vol. 10, 307-315, May 1999
© 1999 American Association for Cancer Research

Phorbol Ester-induced Mononuclear Cell Differentiation Is Blocked by the Mitogen-activated Protein Kinase Kinase (MEK) Inhibitor PD980591

Hongchin He, Xiantao Wang, Myriam Gorospe, Nikki J. Holbrook and Michael A. Trush2

Division of Toxicological Sciences, Department of Environmental Health Sciences, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Maryland 21205 [H. H., M. A. T.], and Gene Expression and Aging Section, National Institute on Aging, NIH, Baltimore, Maryland 21224 [X. W., M. G., N. J. H.]


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The purpose of this study was to evaluate whether the mitogen-activated protein kinase (MAPK) signaling pathway contributes to 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mononuclear differentiation in the human myeloblastic leukemia ML-1 cells. Upon TPA treatment, the activity of ERK1 and ERK2 rapidly increased, with maximal induction between 1 and 3 h, while ERK2 protein levels remained constant. The activity of JNK1 was also significantly induced, with JNK1 protein levels increasing moderately during exposure to TPA. Treatment of cells with PD98059, a specific inhibitor of mitogen-activated protein kinase kinase (MEK), inhibited TPA-induced ERK2 activity. Furthermore, PD98059 completely blocked the TPA-induced differentiation of ML-1 cells, as assessed by a number of features associated with mononuclear differentiation including changes in morphology, nonspecific esterase activity, phagocytic ability, NADPH oxidase activity, mitochondrial respiration, and c-jun mRNA inducibility. We conclude that activation of the MEK/ERK signaling pathway is necessary for TPA-induced mononuclear cell differentiation.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cell differentiation appears to be mediated by an orchestrated series of genetically controlled events (1 , 2) . The transcription factor AP-1,3 composed of members of the Jun and/or Fos families of proteins, recognizes and binds to specific DNA sequences and stimulates transcription of genes responsive to diffentiation-inducing agents (3 , 4) . It has been demonstrated that expression of c-jun and c-fos, as well as AP-1 activity, were induced during TPA-induced monocyte/macrophage differentiation of the human myeloid leukemia cell lines HL-60 and U937 (5, 6, 7) .

The activity of AP-1 is regulated largely through its phosphorylation by members of the MAPK (8, 9, 10, 11) . MAPKs constitute a superfamily of three related kinases that are activated by a diverse array of extracellular stimuli (12, 13, 14) . They include the ERKs (15) , the JNKs (16 , 17) , and p38 (18, 19, 20) . ERK, JNK, and p38 can all be activated by a variety of stimuli, but the kinases are differentially affected by certain signals. For example, ERKs are most highly activated in response to mitogenic stimulation, whereas JNKs and p38 show greater activation in response to cellular stress (14) .

The pathway leading to ERK activation by growth factors and other mitogens has been studied extensively. The first step involves activation of membrane-associated tyrosine kinases, followed by the sequential activation of Ras and Raf (2 , 21 , 22) . Raf then phosphorylates the MEK (23) , which in turn activates ERK (24 , 25) . Importantly, some treatments, such as TPA, appear to bypass the first two steps, entering into the pathway at the level of Raf through a PKC-dependent pathway (26, 27, 28) . The JNK and p38 kinases are activated through analogous pathways but require different MEKs for their phosphorylation and involve distinct upstream mediators (14) . The initiating events involved in triggering the activation of these phosphorylation cascades are poorly understood.

Activation of the MAPK cascades has been demonstrated to be sufficient and necessary for both neuronal differentiation (29 , 30) and megakaryocytic differentiation (31) . MAPK signaling pathway also appears to be involved in the mononuclear differentiation of U937 cells, where ERK and JNK were shown to be activated upon treatment with TPA (10) . Accordingly, expression of constitutively active MEK1 in U937 cells stimulated JNK activity and AP-1-mediated transcriptional activity (32) . However, although activation of MEK1 was sufficient to inhibit cell growth, it did not induce the differentiation of U937 cells into macrophages (32) . In contrast, recent studies showed that the total ERK activity was dramatically reduced during TPA-induced HL-60 cell differentiation (33) . Thus, the exact biological function of the MAPK cascades in mononuclear cell differentiation is not clear.

In this study, we have investigated the importance of the ERK signaling pathway in another model of mononuclear cell differentiation, that of the human myeloblastic leukemic cell line ML-1. Preliminary studies in this laboratory showed that ML-1 cells more readily undergo monocytic differentiation in response to TPA than HL-60 (data not shown). It has also been shown that almost 50 times less TPA is required to induce the differentiation of ML-1 cells than for the differentiation of HL-60 cells (34) . Thus, the differentiation of ML-1 cells is a valuable in vitro system to study the development of phenotypes of monocytes/macrophages at various stages of cell differentiation. The ML-1 cell line was established from the peripheral blood of a patient with acute myeloblastic leukemia (35) . Upon treatment with DMSO or TPA, ML-1 cells undergo granulocytic or monocytic cell differentiation, respectively (36, 37, 38, 39) . Because these cells differentiate along the mononuclear pathway, they exhibit features typical of macrophages, including morphology, nonspecific esterase activity, NADPH oxidase activity, specific cell surface antigens CD11b and CD14, phagocytic ability, and mitochondrial respiration (36, 37, 38, 39) . Here, we provide evidence that ERK activation is required for TPA-induced mononuclear differentiation of ML-1 cells.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Induction of ERK1, ERK2, and JNK1 by TPA in ML-1 cells.
TPA has been shown to be a potent activator of both ERK and JNK (8, 9, 10) . To determine whether ERK and JNK were stimulated during the differentiation of ML-1 cells into monocytes/macrophages, the activity of these kinases was quantitated at various times after treatment with 0.3 ng/ml TPA. ERK2 was immunoprecipitated from cell lysates and assayed for kinase activity by measuring its ability to phosphorylate MBP. As shown in Fig. 1ACitation , the activity of ERK2 rapidly increased in response to TPA with a 6-fold and 4-fold induction at 1 and 3 h, respectively. It then decreased to below the basal levels thereafter (Fig. 1A)Citation . The time-dependent activation of ERK2 was further illustrated by a shift to a slower-migrating form of ERK2, as observed by Western blot analysis, representing the phosphorylated, and therefore activated, ERK2 (Fig. 1B)Citation . To ascertain if the gel mobility-retarded bands at 1 and 3 h were the phosphorylated and activated form of ERK2 and to evaluate the participation of ERK1 in differentiation, Western blot analysis was performed using an antibody recognizing only the dually phosphorylated and activated ERK1 and ERK2. Indeed, both ERK1 and ERK2 were phosphorylated, exhibiting maximal phosphorylation at 1–3 h (Fig. 1C)Citation , whereas total ERK2 protein levels remained constant throughout the TPA treatment period (Fig. 1B)Citation . The activation of the JNK after TPA treatment of ML-1 cells was then examined. JNK1 was immunoprecipitated from cell lysates, and its activity was determined by assessing the ability of the immunoprecipitates to phosphorylate an exogenous substrate, GST-c-Jun (1-135). As shown in Fig. 2ACitation , exposure to TPA led to a significant time-dependent increase in JNK1 activity. Western blot analysis indicated that the total levels of JNK1 protein increased slightly by treatment with TPA (Fig. 2B)Citation . Although the moderate elevation of JNK1 protein level may partly account for the elevation in JNK1 kinase activity (Fig. 2A)Citation , it is likely to be insufficient to mediate the dramatic enhancement in JNK1 kinase activity.



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Fig. 1. Induction of ERK by TPA. A, kinetics of ERK2 activation by TPA. After treatment of ML-1 cells with 0.3 ng/ml TPA for the indicated times, cellular protein extracts were isolated and immunoprecipitated using an antibody against ERK2 as described in "Materials and Methods." ERK2 activity in the immune complex was measured by quantitating the incorporation of 32P onto MBP after resolving the kinase assay reactions by electrophoresis in 12% SDS-polyacrylamide gels. B, Western blot analyses of ERK2 protein expression after treatment with TPA. Cellular protein extracts were prepared and subjected to electrophoresis and blotting as described in "Materials and Methods." The blots were hybridized with an antibody recognizing total ERK2. C, Western blot analysis of ERK1 and ERK2 activation. Blots were hybridized with Anti-Active MAPK antibody, which specifically recognizes the phosphorylated and activated forms of ERK1 and ERK2.

 


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Fig. 2. Time course of JNK1 activation and protein expression in response to TPA. A, after treatment of ML-1 cells with 0.3 ng/ml TPA for the indicated times, JNK1 activity was determined by an immune complex kinase assay using GST-c-Jun as a substrate. B, Western blot analysis of JNK1 protein expressions in ML-1 cells at the indicated times after treatment with 0.3 ng/ml TPA.

 
Effect of the Specific MEK Inhibitor PD98059 on TPA-mediated ERK2 Activation.
The compound PD98059 has been shown to selectively block MEK activation and, consequently, the activation of the ERK without affecting the JNK or p38 signaling pathways (40 , 41) . Consistent with this specificity, treatment of PC12 cells with PD98059 was shown to inhibit MEK and ERK activations by nerve growth factor and to block neuronal differentiation (42) . PD98059 has also been shown to similarly prevent megakaryocytic differentiation of human erythroleukemia K562 cells induced by TPA (31) . To examine whether the activation of ERK following exposure to TPA was important for mononuclear cell differentiation, we used PD98059 to pharmacologically block this pathway. As shown in Fig. 3Citation , exposure to PD98059 led to a 2-fold and a 10-fold reduction in TPA-activated ERK2 activity at 1 and 3 h, respectively. PD98059 also inhibited the basal level of ERK2 activity in ML-1 cells (Fig. 3)Citation . Treatment of the cells with PD98059 had no significant effect on TPA-induced JNK1 activity (Fig. 4)Citation .



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Fig. 3. Inhibition of TPA-induced ERK2 activity by the MEK inhibitor PD98059. Cellular protein extracts were isolated after addition of 0.3 ng/ml TPA to ML-1 cells in the absence or presence of 10 µM PD98059 for the indicated time. Endogenous ERK2 was immunoprecipitated, and the kinase activity associated with the immune complex was assayed using substrate MBP as described in "Materials and Methods."

 


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Fig. 4. Effect of PD98059 on JNK1 activity. Cellular protein extracts were isolated after addition of 0.3 ng/ml TPA to ML-1 cells in the absence or presence of 10 µM PD98059 for the indicated time. Endogenous JNK1 was immunoprecipitated, and the kinase activity associated with the immune complex was assayed using GST-c-Jun as a substrate.

 
Expression of c-jun and c-fos mRNA in Response to TPA Treatment: Effect of PD98059.
Phorbol ester-induced cell differentiation is mediated, at least in part, by the activation of the AP-1 complex (1 , 3) . Therefore, we explored the expression of c-jun and c-fos during TPA-induced differentiation of ML-1 cells. The kinetics of induction of c-jun and c-fos mRNAs in ML-1 cells in response to 0.3 ng/ml TPA was assessed by Northern blot analysis. As shown in Fig. 5Citation , TPA treatment of ML-1 cells led to a elevation of c-jun mRNA, which reached a maximum by 6 h. By contrast, c-fos mRNA was rapidly induced within 1 h exposure to TPA and remained relatively constant for at least 24 h (Fig. 5)Citation .



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Fig. 5. Effect of PD98059 on c-jun and c-fos mRNA expression. Total RNA was isolated from cells treated with TPA in the absence or presence of 10 µM PD98059 for the indicated times and subjected to Northern blot analysis. Blots were stripped and reprobed with an 18S rRNA probe to visualize differences in loading and transfer among samples. Blots shown are representative of two independent experiments.

 
Because ERK and JNK have been proposed to regulate AP-1 function (9) , we examined the effect of PD98059 on the expression of c-jun and c-fos mRNA in response to TPA. As shown in Fig. 5Citation , TPA-induced c-jun mRNA expression was dramatically inhibited by treatment with PD98059, whereas that of c-fos mRNA was affected minimally.

Effect of PD98059 on TPA-induced ML-1 Cell Differentiation.
Exposure to TPA induces macrophage-like differentiation of ML-1 cells (36, 37, 38, 39) . To evaluate the contribution of ERK in this process, PD98059 was used to examine the biological consequences of inhibiting the MEK/ERK signaling pathways. ML-1 cells treated with 0.3 ng/ml TPA for 3 days, followed by an additional 3 days in culture after the removal of TPA, displayed morphological features typical of monocytes/macrophages (Fig. 6c)Citation as they ceased to proliferate and were enlarged, with a higher cytoplasmic:nuclear ratio than that of undifferentiated ML-1 cells (Fig. 6, a and c)Citation . By contrast, cells treated simultaneously with 10 µM PD98059 and 0.3 ng/ml TPA continued to proliferate and exhibited a morphology resembling that of undifferentiated cells (Fig. 6, a and d)Citation . PD98059 itself did not exert any detectable effect on either cell growth or differentiation (Fig. 6b)Citation .



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Fig. 6. Effect of PD98059 on TPA-induced morphological characteristics of ML-1 cells. a, undifferentiated ML-1 cells; b, cells treated with 10 µM PD98059 alone; c, cells treated with 0.3 ng/ml TPA for 3 days, followed by an additional 3 days in culture without TPA; d, cells differentiated with 0.3 ng/ml TPA in the presence of 10 µM PD98059 for 3 days, followed by an additional 3 days in the absence of TPA and PD98059. x200.

 
The effect of PD98059 on differentiation was assessed by studying the expression of the monocyte/macrophage-specific marker {alpha}-naphthyl acetate esterase (NSE). As shown in Fig. 7ACitation (panel c), cells differentiated with 0.3 ng/ml TPA displayed positive NSE staining, exhibiting red-brown cytoplasms. NSE staining of cells treated with 0.3 ng/ml TPA plus 10 µM PD98059 was similar to that of undifferentiated ML-1 cells (Fig. 7ACitation , panels a and d). The inhibition of the expression of NSE activity by PD98059 was dose dependent. Ninety % of the TPA-differentiated cells were NSE-positive, whereas only 20 or 15% of the cells treated with TPA plus 10 or 20 µM PD98059 were NSE positive (Fig. 7B)Citation .



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Fig. 7. Effect of PD98059 on ML-1 cell NSE activity. A, representative microscopic appearance of the cells stained for NSE activity. a, undifferentiated ML-1 cells; b, cells treated with 10 µM PD98059 alone; c, cells treated with 0.3 ng/ml TPA for 3 days, followed by an additional 3 days in culture without TPA; d, cells differentiated with 0.3 ng/ml TPA in the presence of 10 µM PD98059 for 3 days, followed by an additional 3 days in culture without TPA and PD98059. B, cells were treated with 0.3 ng/ml TPA and the indicated doses of PD98059 for 3 days, followed by an additional 3 days in culture in the absence of either compound. Undiff., undifferentiated ML-1 cells. A minimum of 200 cells/slide were counted, and percentages of brown-stained positive cells were calculated. Values presented are means; bars, SE; n = 3.

 
We then assessed the effect of PD98059 on the ability of the cells to perform phagocytosis, another marker of macrophage differentiation. Compared with undifferentiated cells (untreated cells in Fig. 8ACitation , panel a), TPA-differentiated cells were able to effectively ingest opsonized SRBCs, as shown in Fig. 8ACitation (panel c). In contrast, neither cells treated with PD98059 alone nor cells treated with TPA plus PD98059 were phagocytic, as evidenced by the presence of SRBCs outside the cells (Fig. 8ACitation , panels b and d, respectively). As shown in Fig. 8 (B and C)Citation , both the percentage of phagocytic cells and the number of SRBCs ingested per cell were decreased by treatment with PD98059.



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Fig. 8. Effect of PD98059 on phagocytosis of SRBCs by ML-1 cells. A, representative photomicrographs of ML-1 cells analyzed for phagocytic capability as described in "Materials and Methods." a, undifferentiated ML-1 cells; b, cells treated with 10 µM PD98059 alone; c, cells differentiated with 0.3 ng/ml TPA for 3 days, followed by an additional 3 days in culture without TPA; d, cells differentiated with 0.3 ng/ml TPA in the presence of 10 µM PD98059 for 3 days, followed by an additional 3 days in the absence of TPA and PD98059. x200. B, percent cells phagocytic. C, number of SRBCs ingested per ML-1 cell. Values presented are means; bars, SE; n = 3.

 
Superoxide produced by NADPH oxidase is another marker used frequently to monitor granulocytic and monocytic differentiation because the undifferentiated cells do not express NADPH oxidase activity (43, 44, 45) . Therefore, we measured the ability of ML-1 cells to produce superoxide via NADPH oxidase both at a basal level (resting state) and at a state stimulated by exposure the cells to 30 ng/ml TPA in the assay reactions. Undifferentiated ML-1 cells showed little, if any, superoxide production (data not shown), whereas cells differentiated with TPA in the absence of PD98059 exhibited a basal level of 6.5 nmol superoxide/h/106 cells (Fig. 9A)Citation and a stimulated level of 70 nmol superoxide/h/106 cells (Fig. 9B)Citation . In cells treated with 0.3 ng/ml TPA in the presence of various doses of PD98059, the basal level of superoxide production decreased in a dose-dependent manner (Fig. 9A)Citation . PD98059, at 10 µM concentration, decreased the superoxide production down to that of undifferentiated cells (Fig. 9A)Citation . PD98059 also caused a dose-dependent reduction in superoxide production by the stimulated NADPH oxidase in cells differentiated with TPA with the exception of that at 2.5 µM concentration of PD98059 (Fig. 9B)Citation . Of interest was the finding that cells differentiated in the presence of TPA plus 2.5 µM PD98059 exhibited a greater superoxide production than cells terminally differentiated with TPA in the absence of PD98059. The cells treated with TPA plus 2.5 µM PD98059 continued to proliferate and, morphologically, they appeared to be less mature than the TPA-differentiated cells (data not shown). Other investigators have previously observed that less differentiated monocytes have a higher ability to generate superoxide via NADPH oxidase than do mature macrophages (36 , 46 , 47) .



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Fig. 9. Effect of PD98059 on NADPH oxidase activity in TPA-differentiated ML-1 cells. ML-1 cells were treated with 0.3 ng/ml TPA and indicated doses of PD98059 for 3 days, followed by 3 days in culture after the removal of both TPA and PD98059. A, basal level of superoxide produced by NADPH oxidase at a resting state. B, superoxide produced by NADPH oxidase at a stimulated state. Values presented are means; bars, SE; n = 3.

 
Finally, we assessed the effect of PD98059 on mitochondrial maturation during TPA-induced ML-1 cell differentiation. Fully differentiated macrophages exhibit mitochondrial respiration, whereas undifferentiated myeloid cells, monocytes, and granulocytes lack appreciable levels of mitochondrial respiration (39 , 48 , 49) . As ML-1 cells differentiate into macrophages, they exhibit increases in mitochondrial-dependent oxygen consumption and ATP production, which can be inhibited by the mitochondrial protein synthesis inhibitor chloramphenicol (39) . Superoxide produced as a by-product of mitochondrial respiration can be measured by using a chemilumigenic probe, lucigenin, and it has been shown to be a sensitive biomarker of mitochondrial maturation during ML-1 cell differentiation (50 , 51) . As shown in Fig. 10Citation , mitochondrial superoxide production in the TPA-differentiated cells was inhibited by PD98059 in a dose-dependent manner.



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Fig. 10. Effect of PD98059 on mitochondrial respiration in TPA-differentiated cells. ML-1 cells were treated with 0.3 ng/ml TPA and indicated doses of PD98059 for 3 days, followed by 3 days in culture after the removal of both. Superoxide production via mitochondrial respiration was assessed by LDCL. Values presented are means; bars, SE; n = 3.

 
Taken together, treatment of PD98059 abolished TPA-induced mononuclear cell differentiation of ML-1 cells as assessed by cell morphology, NSE activity, phagocytic ability, NADPH oxidase activity, and mitochondrial respiration. These findings strongly indicate a role for ERK in mononuclear differentiation of ML-1 cells.


    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The TPA-induced mononuclear differentiation of human myelogenous leukemia cells is believed to be mediated through PKC (52, 53, 54, 55) . The biological activity of TPA is initiated by its binding to PKC, followed by the translocation of PKC from the cytoplasm to cellular membranes, resulting in its activation (56 , 57) . A recent report has shown that TPA-induced differentiation of HL-60 cells was inhibited by a PKC-specific inhibitor, suggesting that PKC is essential for TPA-induced HL-60 cell differentiation (58) .

Much evidence supports the involvement of the ERK cascade as an important signaling pathway in cell differentiation. In this regard, neuronal differentiation of PC12 cells is one of the most extensively studied systems. Constitutively active mutants of MEK and active forms of each component of the ERK cascade have been used to demonstrate that persistent activation of MEK or ERK is sufficient for the differentiation of PC12 cells (29 , 30) . Conversely, expression of dominant-negative MEK mutants that prevent ERK activation or expression of a phosphatase that inactivates ERK abolish the differentiation-inducing effect of MEK kinase and MEK (29 , 30) . Moreover, the nerve growth factor-induced neurite formation of PC12 cells is blocked by the specific MEK inhibitor PD98059, providing additional evidence that the MEK/ERK pathway is necessary for PC12 cell differentiation (42) .

The essential role of ERK cascade in the differentiation of other cell types has also been demonstrated. During T-cell maturation, the positive selection of thymocyte development was shown to be blocked when a catalytically inactive form of MEK1 was expressed in immature cells (59 , 60) . Recently, Whalen et al. (31) demonstrated that ERK was activated during the TPA-induced megakaryocytic differentiation of K562 cells. Using constitutively active MEK mutants and PD98059, they showed that the activation of ERK and MEK was sufficient and necessary for megakaryocytic differentiation (31) . In other studies, TPA-induced differentiation of U937 cells into monocytes/macrophages was also accompanied by activation of ERK1 and ERK2 (10) . Raf and ERK were reportedly activated during mononuclear differentiation of HL-60 cells induced by TPA (61) . These findings are in agreement with our observations reported here that ERK1, ERK2, and JNK1 were activated during the TPA-induced mononuclear differentiation of ML-1 cells (Figs. 1Citation and 2)Citation . By contrast, a recent report by Meighan-Mantha et al. (33) showed that, during TPA-induced differentiation of HL-60 cells, total ERK activity was dramatically reduced, an effect that was attributed primarily to the rapid deactivation of ERK1. The discrepancies between this study and ours may perhaps reflect differences in the cell lines used. However, similar to the findings of Meighan-Mantha et al. (33) , we also observed that ERK1 and ERK2 activities were reduced to below basal levels after a rapid early induction (Fig. 1)Citation . It is not clear at present whether this reduction in ERK1 and ERK2 activity at later times plays any role in mononuclear cell differentiation.

The biological effects of TPA are mediated, at least in part, by the generation of active AP-1 complexes, which are predominantly composed of heterodimers of c-Jun/c-Fos. Activation of the c-Jun/c-Fos AP-1 complexes is often mediated by phosphorylation of the protein components, thus resulting in enhanced AP-1 function (8, 9, 10, 11) . Our data presented here indicate an additional level of regulation wherein ERK potentially regulates AP-1 activity by directly altering c-jun mRNA levels. The experiments using PD98059 indicate that ERK-regulated c-jun mRNA expression by TPA is specific, because addition of PD98059 completely suppresses TPA-induced c-jun mRNA but only minimally affects the TPA-mediated induction of c-fos mRNA (Fig. 5)Citation . Thus, during TPA-induced differentiation, AP-1 activity may be influenced by ERK (and/or JNK)-mediated phosphorylation of c-Jun and c-Fos, but additionally, ERK also tightly regulates c-jun mRNA expression. Whether c-jun expression directly influences the differentiation phenotype remains to be determined.

In summary, we have provided evidence supporting the requirement for MEK/ERK activation in the induction of differentiation of ML-1 cells by TPA. The specific MEK inhibitor PD98059 blocked the activation of ERK subfamily of MAPK, expression of TPA-induced c-jun mRNA, and mononuclear differentiation of ML-1 cells, as assessed by a spectrum of parameters: cellular morphology (Fig. 6)Citation , NSE activity (Fig. 7)Citation , phagocytic capability (Fig. 8)Citation , NADPH oxidase activity (Fig. 9)Citation , and mitochondrial maturation (Fig. 10)Citation . Whether MEK/ERK activation is sufficient for differentiation is yet to be determined. This study not only provides insight into the molecular mechanisms that underlie the process of macrophage differentiation, but it also supports the concept that the MEK/ERK signaling pathway plays an important role in the differentiation of a number of cell types.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Chemicals and Materials.
RPMI-1640, penicillin, streptomycin, and Dulbecco’s PBS were purchased from Life Technologies, Inc., (Grand Island, NY). FBS and all other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Sheep blood was purchased from Becton Dickinson (Cockeysville, MD). IgG antibody against sheep erythrocytes was purchased from Diamedix (Miami, FL).

Cell Culture.
The myeloblastic leukemic cell line ML-1 was kindly provided by Dr. Ruth Craig (Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH). ML-1 cells were maintained in RPMI 1640 supplemented with 7.5% heat-inactivated FBS. ML-1 cells were grown at 37°C in a humidified atmosphere of 5% CO2. For experiments in which the cells were induced to differentiate, they were seeded in FBS-coated flasks at a density of 3 x 105 cells/ml (37 , 39) . Differentiation was induced by treatment of cells with 0.3 ng/ml TPA (dissolved in DMSO; final concentration in medium, 0.03%) for 3 days, whereupon TPA-containing medium was then removed and replaced with fresh medium lacking TPA, and the cells were incubated for an additional 3 days. In experiments using the MEK inhibitor PD98059 (Calbiochem, La Jolla, CA), cells were simultaneously treated with 0.3 ng/ml TPA, and various concentrations of PD98059 (dissolved in DMSO; final concentration in medium, 0.03%) for 3 days. Medium was then removed and replaced with fresh medium without either TPA or PD98059, and cells were cultured for an additional 3 days. Cell viability was determined by trypan blue exclusion after the cells were harvested from the flasks and resuspended in complete Dulbecco’s PBS with 0.01% MgCl2, 0.01% CaCl2, and 0.1% glucose at a concentration of 1 x 106 cells/ml. For morphological characterization, photomicrographs were taken using an inverted phase contrast microscope while the cells were in the tissue culture flasks.

Mitochondrial-dependent LDCL.
LDCL was used to assess the production of superoxide anion from mitochondrial respiration (50) . Briefly, cells were harvested and resuspended in Dulbecco’s PBS. One million cells were used in a total volume of 2.5 ml. The final concentration of lucigenin (bis-N-methylacridinium nitrate; Aldrich, Milwaukee, WI) was 1 µM, a concentration at which lucigenin does not redox-cycle (51) . LDCL was performed using a Berthold Biolumat LB9505 at 37°C for 1 h. Data from LDCL experiments is expressed as integrated chemiluminescence.

Superoxide Anion Production by NADPH Oxidase.
The superoxide dismutase-inhibitable reduction of cytochrome c was used to quantitate production of superoxide anion generated by NADPH oxidase. This protocol was modified from the method described by Kensler and Trush (62) . The reactions contained 106 cells and 3 mg cytochrome c in a final volume of 2 ml, either containing or lacking 0.15 mg of superoxide dismutase. After incubation for 1 h at 37°C, the reactions were terminated by placing tubes at 4°C, followed by centrifugation. The supernatants were assayed spectrophotometrically at 550 nm. Absorbance values were converted to nmol superoxide production/h/106 cells. The values were corrected for the portion of nonsuperoxide-dependent reduction of cytochrome c, which was not inhibitable by superoxide dismutase. For measurement of NADPH oxidase activity at a stimulated state, TPA (final concentration, 30 ng/ml) was used in the assay reactions.

NSE Activity.
The analysis of nonspecific esterase of the cells was carried out using {alpha}-naphthyl acetate as a substrate essentially as described by Ennist and Jones (63) . Cells were harvested and resuspended in complete PBS at 106 cells/ml. The working buffer-substrate solution, made immediately before use, consisted of 10 ml of 0.1 M phosphate buffer (pH 7.3), 0.25 ml 1% {alpha}-naphthyl acetate, and 0.8 ml of freshly made hexazonium pararosaniline. One ml of buffer-substrate solution was added to 1 ml of cell suspension and incubated at room temperature for 5 min. Cells were then washed with PBS, and 300 cells were counted; cells displaying intense red-brown azo dye in the cytoplasm were scored as NSE positive.

Fc Membrane Receptor-mediated Phagocytosis.
A 0.2-ml aliquot of a 0.25% suspension of opsonized SRBCs in RPMI 1640 was added to 0.5 x 106 cells in a final volume of 0.7 ml. Then the Fc membrane receptor-mediated phagocytic assay was carried out in a humidified incubator filled with 5% CO2 in air at 37°C for 30–45 min. Noningested RBCs were hypotonically lysed for 30 s by adding distilled water. The hypotonic condition was restored to isotonicity with an equal volume of 1.8% NaCl. Cells were then washed with PBS and resuspended in 1 ml of PBS. Slides were prepared by using a cytospin (Shandon) at 1000 rpm for 5 min. The slides were air-dried, fixed with methanol, and stained with Wright-Giemsa (Fisher). The stained cells were visualized microscopically at a magnification of x400 to quantify the percentage of cells containing SRBCs and the number of SRBCs ingested per actively phagocytic cells. At least 100 cells/slide were counted.

Northern Blotting Analysis.
Total RNA was isolated using the procedure initially described by Chomczynski and Sacchi (64) and Qiagen RNeasy Mini kit (Qiagen, Inc., Chatsworth, CA). RNA was electrophoresed on 1% agarose gels containing formaldehyde and transferred to a Nytran membrane (Schleicher & Schuell, Keene, NH). The cDNA probes were labeled with [{alpha}-32P]dCTP (ICN, Costa Mesa, CA) using a random primer labeling kit (Boehringer Mannheim, Mannheim, Germany). Membranes were hybridized with the cDNA probe overnight at 42°C in a buffer containing 6x SSC, 50% formamide, 5x Denhardt’s reagent, and 0.5% SDS. After the membranes were washed with 1x SSC and 0.1% SDS, they were subjected to autoradiography at -70°C with an intensifying screen and/or quantitated by PhosphorImager analysis. An oligomer complementary to 18S rRNA was end-labeled using T4 polynucleotide kinase (Life Technologies, Inc.) and used to normalize for differences in loading and transfer among samples.

Western Blotting Analysis.
Whole-cell extracts were prepared by lysing 107 cells with buffer containing 20 mM HEPES (pH 7.4), 2 mM EGTA, 50 mM ß-glycerophosphate, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 1% Triton X-100, 10% glycerol, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, and 1x Complete (protease inhibitor cocktail; Boehringer Mannheim). Protein extracts were then prepared by centrifugation at 10,000 x g for 10 min at 4°C. Protein concentration in each supernatant was determined by using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Richmond, CA). Fifty to 100 µg of total protein were resolved on 12% SDS-PAGE gels and transferred onto Immobilon-P membrane (Millipore, Bedford, MA). The membranes were blocked with 4% nonfat dry milk in PBS-T (0.05% Tween 20 in PBS) for 1 h. After washing with three changes of PBS-T for 30 min, the membranes were incubated with a primary antibody for 1 h at room temperature. The primary antibodies used were monoclonal antibody specific for ERK2 (Transduction Laboratories, Lexington, KY), polyclonal antibody Anti-Active MAPK specific for the dually phosphorylated and activated form of ERKs (Promega Corp., Madison, WI), and polyclonal antibody specific for JNK1 (Santa Cruz Biotechnology, Santa Cruz, CA). The membranes were then incubated with a horseradish peroxidase-conjugated secondary antibody and developed using an enhanced chemiluminescence (ECL) assay system (Amersham). The density of the bands was quantitated using a densitometer (Molecular Dynamics).

Kinase Activity Assays.
Kinase activity was measured after immunoprecipitation of endogenous JNK1 and ERK2 according to Guyton et al. (65) . Briefly, cells were harvested and washed with ice-cold PBS and then lysed in a buffer containing 20 mM HEPES (pH 7.4), 2 mM EGTA, 50 mM ß-glycerophosphate, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 1% Triton X-100, 10% glycerol, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, and 1x Complete (protease inhibitor cocktail; Boehringer Mannheim). Protein extracts were then cleared of cellular debris by centrifugation at 10,000 x g for 10 min at 4°C. Cellular ERK2 and JNK1 were immunoprecipitated from the extract using rabbit polyclonal antibodies against p42ERK2 or p46JNK1 (Santa Cruz Biotechnology). Kinase activity was assayed for 20 min at 30°C in the presence of 30 µM ATP, 20 µCi of [{gamma}-32P]ATP, and 6 µg of substrate in assay buffer containing 20 mM MOPS (pH 7.2), 2 mM EGTA, 20 mM MgCl2, and 0.1% Triton X-100. The substrate used for measuring ERK2 activity was MBP, and the substrate used for measuring JNK1 activity was GST-c-Jun (1-135). The reactions were terminated by addition of Laemmli buffer and boiling samples and were resolved by SDS-PAGE in 12% gels. Gels were then dried and subjected to autoradiography and/or quantitated by PhosphorImager analysis. Kinase activity was determined by measuring the amount of 32P incor-porated onto the substrates.


    Acknowledgments
 
We thank Dr. Thomas Primiano for critical review of the manuscript. We are grateful to Dr. Eva Szabo for human c-jun cDNA probe, Dr. Daniel Nathans for human c-fos cDNA probe, and Dr. Jim Woodgett for GST-c-jun (1-135) construct.


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

1 This work was supported by Grants ES03760, ES07141, ES08078, and ES03819 from the National Institute of Environmental Health Sciences, NIH. Back

2 To whom requests for reprints should be addressed, at Division of Toxicological Sciences, Department of Environmental Health Sciences, School of Hygiene and Public Health, The Johns Hopkins University, 615 North Wolfe Street, Baltimore, MD 21205. Phone: (410) 955-4712; Fax: (410) 955-0116. Back

3 The abbreviations used are: AP-1, activator protein-1; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; MEK, ERK kinase; TPA, 12-O-tetradecanoylphorbol-13-acetate; PKC, protein kinase C; GST, glutathione S-transferase; NSE, nonspecific esterase; SRBC, sheep RBC; FBS, fetal bovine serum; LDCL, lucigenin-derived chemiluminescence; MBP, myelin basic protein. Back

Received for publication 1/12/98. Revision received 3/ 9/98. Accepted for publication 6/10/98.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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