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[Cancer Research 64, 5425-5433, August 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Up-Regulation of Egr1 by 1,25-Dihydroxyvitamin D3 Contributes to Increased Expression of p35 Activator of Cyclin-Dependent Kinase 5 and Consequent Onset of the Terminal Phase of HL60 Cell Differentiation

Fei Chen, Qing Wang, Xuening Wang and George P. Studzinski

Department of Pathology and Laboratory Medicine, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Advances in differentiation therapy of cancer are likely to depend on improved understanding of molecular events that underlie cell differentiation. We reported recently that cyclin-dependent kinase (Cdk)5 and p35Nck5a (p35) are expressed in human leukemia HL60 cells induced to differentiate to monocytes by an exposure to 1,25-dihydroxyvitamin D3 (1,25D3), form a complex, and this complex has kinase activity (F. Chen and G. P. Studzinski, Blood 2001;97:3763). This laboratory has also provided evidence that the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway is active in the early (24–48 h) stages of HL60 cell differentiation induced by 1,25D3 but declines in the later, terminal phase of this form of differentiation (X. Wang and G. P. Studzinski, J Cell Biochem 2001;80:471). We examine now the hypothesis that Egr1 protein contributes to the up-regulation of p35 gene transcription and, thus, activated Cdk5/p35 kinase phosphorylates and inactivates mitogen-activated protein/extracellular signal-regulated kinase kinase 1 (MEK1). Our data show that in 1,25D3-treated cells, p35 and Egr1 protein levels are elevated in a dose-dependent manner at the onset of the late stage of differentiation. We show also that 1,25D3 treatment of HL60 cells markedly increases the binding of Egr1 to an element in the p35 gene promoter, whereas transfection of an excess of this Egr1-binding oligonucleotide ("promoter decoy") reduces p35 gene transcription and cell differentiation. Additionally, Cdk5/p35 phosphorylates MEK1 and inhibits its ability to phosphorylate its downstream target Erk2. These data suggest that in 1,25D3-treated HL60 cells, Egr1 up-regulates p35 gene transcription and that Cdk5/p35 kinase inactivates the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway by phosphorylation of MEK1, and this contributes to terminal differentiation of these cells.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The 1,25-dihydroxyvitamin D3 (1,25D3), a metabolite and an active form of vitamin D3, is a ligand for vitamin D receptor, a member of the nuclear receptor family. After it is liganded by 1,25D3, the receptor forms homodimers or heterodimers with retinoid X receptor subtypes, which regulate gene expression by binding to vitamin D response elements in the promoter regions of target genes (1, 2, 3) . Although a number of genes have been identified that are directly regulated by 1,25D3 through the vitamin D response elements in their promoters and are involved principally in bone and calcium metabolism, regulation of monocytic differentiation by 1,25D3 is less well understood. However, at least some of the direct 1,25D3 response genes appear to activate intracellular signaling networks, including the mitogen-activated protein kinase (MAPK) pathways, perhaps as an indirect consequence of their increased expression (4, 5, 6) .

The MAPK pathways have an important position in the network of interactive signaling cascades in mammalian cells (7) . To a large extent, the activation and intracellular localization of the constituent kinases are controlled by the formation of complexes with other kinases and/or nonkinase scaffolding proteins and may be regulated positively or negatively by many factors, including interactions with components of other signaling pathways (8, 9, 10, 11) . For instance, it has been reported recently that in neuronal cells of mice, activated cyclin-dependent kinase (Cdk)5 phosphorylates and inactivates mitogen-activated protein/extracellular signal-regulated kinase kinase 1 (MEK1) on Thr286 in vivo as well as Ras-activated MEK1 in vitro (12) . The Thr286 phosphorylation of MEK1 by Cdk5 results in the inhibition of catalytic activity of MEK1 and consequently of the phosphorylation of extracellular signal-regulated kinase (Erk)1/2 in vitro (12) . It has been shown also that in rat PC12 cells, sustained activation of Erk induces the transcription factor Egr1, which up-regulates p35 transcription and thereby activates Cdk5 (13) . Thus, because Egr1 appears to be a downstream target of Erk1/2 (13) , it is possible that Cdk5/p35 can act as a feedback regulator or a switch to shut down the MAPK signaling cascade.

Cdk5 is a proline-directed serine/threonine kinase that has sequence homology to Cdks, which regulate cell cycle progression. However, the role of Cdk5 in the control of the cell cycle is not clear (14, 15, 16) . A well-demonstrated role for Cdk5 is the regulation of the cytoarchitecture of neurons in the central nervous system (17, 18, 19, 20, 21) . There are ~2 dozen proteins with diverse functions that have been identified as Cdk5 substrates, and in neurons, this kinase has been implicated in the regulation of microtubule stability and transport, axon guidance, secretion, membrane transport, and dopamine signaling (17, 18, 19, 20, 21) . Recently, several groups, including ours, have demonstrated that Cdk5 also plays an important role in nonneuronal cells and is involved in myogenesis, lens differentiation, spermatogenesis, insulin secretion, and hematopoietic cell differentiation (22, 23, 24, 25, 26, 27, 28, 29) .

We have reported that the expression of Cdk5 and p35 as well as the kinase activity of the complex is required for the differentiation of human promyeloblastic leukemia HL60 cells to mature monocytic cells by exposure to 1,25D3 or for the maturation of normal hematopoietic precursor cells to monocytes (27 , 28) . This laboratory has shown also that the Erk MAPK pathway may contribute to the early stages of HL60 cell differentiation induced by 1,25D3 (4 , 6) . We now present evidence that in HL60 cells, Egr1 is involved in the up-regulation of p35 gene transcription and that Cdk5/p35 kinase can inactivate the Erk MAPK pathway by a phosphorylation of MEK1. This may be responsible for, or contribute to, the 1,25D3-induced cessation of cell proliferation, which is characteristic of the terminal stage of differentiation of these cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Biological Reagents.
HL60-G cells (30) , subcloned from cultures originally derived by Gallagher et al. (31) from a patient with promyeloblastic leukemia, were grown in RPMI 1640 supplemented with 1% L-glutamine (from Mediatech, Washington, DC) and 10% heat-inactivated bovine calf serum (Hyclone, Logan, UT) at 37°C in an environment of 5% CO2. The physiologically active form of vitamin D, 1,25D3, was a kind gift from Dr. Milan Uskokovic (BioXell, Nutley, NJ) and unless otherwise indicated was used at 10 nM concentration. Glutathione S-transferase (GST)-MEK1 and GST-Erk2 were from Upstate Biotechnology Inc. (Upstate Biotechnology Inc., Lake Placid, NY). The antibodies to Cdk5 (DC17), p35 (C190), Egr1 (588), vitamin D receptor (C20), CD14 (M305), cyclin D1 (C-20), Erk1/2 (K-23), Raf1 (C-12), and MEK1 (C-18) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The phospho-Erk1/2 pathway sampler kit, which includes phospho-Raf1 (ser338), phospho-MEK1 (Ser218/222), and phospho-Erk1/2 (Thr202/Tyr204), was purchased from Cell Signaling Technology (Beverly, MA). The antibody for calreticulin was obtained from Santa Cruz Biotechnology.

RNA Isolation and Reverse Transcription-PCR (RT-PCR).
HL60 cells, untreated controls, or cells treated with 1,25D3 were lysed using 1 ml of TRIzol Reagent (Life Technologies, Inc., Grand Island, NY) containing 0.2 ml of chloroform (Sigma, St. Louis, MO). Total cellular RNA was then purified by isopropanol (Sigma) precipitation and washed three times with 70% ethanol. RT-PCR was performed using the RT-PCR kit (Invitrogen, Life Technologies, Inc., Carlsbad, CA) according to the manufacturer’s instructions. The primers used for PCR were as follows: Egr1, upstream primer 5'-AGATGATGCTGCTGAGCAAC-3' and downstream primer 5'-AGTAAATGGGACTGCTGTCG-3'; p35, upstream primer 5'-GCCGTACAGAACAGCAAGAA-3' and downstream primer 5'-GTCGGCATTTATCTGCAGCA-3'; and ß-actin, upstream primer 5'-TGACGGGGTCACCCACACTGTGCCCAGCTA-3' and downstream primer 5'-CTAGAAGCATTTGCCGGTGGACGATGGAGGG-3'.

Western Blotting, Immunoprecipitation, and in Vitro Kinase Reaction Assay.
HL60 cells, untreated controls, or cells treated with 1,25D3 were lysed with 1x cell lysis buffer [20 mM Tris-HCl (pH7.5), 150 mM NaCl, 1 mM Na2 EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium PPI, 1 mM ß-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride; Cell Signaling Technology]. Protein concentrations were determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). Samples for immunoblotting were prepared by mixing aliquots of the protein extracts (30 µg) with 3x SDS sample buffer [150 mM Tris (pH 6.8), 30% glycerol, 3% SDS, bromphenol blue dye 1.5 mg/100 ml, and 100 mM DTT] and denatured by heating at 100°C for 4 min. Protein samples were then separated by SDS-PAGE and transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, NJ). The membrane was subjected to immunoblot analysis, and proteins were visualized by the enhanced chemiluminescence method of detection (Amersham Pharmacia Biotech). The membrane was then stripped and reprobed for calreticulin, a loading control, as described previously (26) . For immunoprecipitation, proteins (200 µg) were incubated with a primary antibody for 2 h at 4°C followed by an incubation with protein A/G PLUS agarose beads for 1 h. Samples were washed four times in 1x cell lysis buffer (Cell Signaling Technology), resuspended in 30 µl of 3x SDS sample buffer, and boiled for 3 min. The proteins were then resolved using a SDS-PAGE gel, transferred to a nitrocellulose membrane, and detected for Western blotting as described previously. For the Raf1-associated in vitro kinase assay, the immunoprecipitated complexes were washed three times with 1x cell lysis buffer then three times with 1x kinase buffer (Cell Signaling). The complexes were then incubated with 2 µg of GST-MEK1 in 30 µl of 1x kinase buffer supplemented with 100 µM ATP for 30 min at 30°C. For the Cdk5-associated kinase assay, the immunoprecipitated complexes were washed three times with 1x cell lysis buffer then 1x kinase buffer. The complexes were then incubated with either GST-MEK1 or Raf1-phosphorylated GST-MEK1 (the supernatant from Raf1-dependent kinase assay) in 30 µl of 1x kinase buffer supplemented with 0.4 mCi/ml [{gamma}32P]ATP (DuPont-NEN, Boston, MA; specific activity, 3000 Ci/mmol), 10 µM ATP for 30 min at 30°C. The reaction was stopped by adding 30 µl of 3x SDS sample buffer, the mixture boiled for 3 min, and the proteins were resolved on a 10% SDS-PAGE gel. The Raf1 phosphorylation of GST-MEK1 was recognized by the specific phospho-MEK1 antibody. The Cdk5-associated phosphorylation of GST-MEK1 was detected by autoradiography of the radioactivity of the 32P-labeled GST-MEK1 on a SDS-PAGE gel.

DNA-Protein Binding ("Gel Mobility Shift") Assays.
Electrophoretic mobility shift assays were performed as follows. The reaction mixture of binding buffer [50 mM KCl, 20 mM HEPES-KOH (pH 7.5), 10 mM MgCl, 10% glycerol, 0.5 mM DTT, and 1% NP40], 0.2 ng of [32{gamma}P]ATP-labeled oligonucleotide, 2 µg poly(deoxyinosinic-deoxycytidylic acid; Pharmacia Biotech, Piscataway, NJ), and 10 µg of nuclear protein, extracted as described previously (4) was incubated at 25°C for 20 min and the reaction products separated on a 4% polyacrylamide gel in 0.25 x Tris-borate EDTA (22.5 mM Tris-borate and 0.5 mM EDTA). For oligonucleotide competition experiments, 10- and 50-fold excess of the unlabeled competitor oligonucleotide was added 1 h before the addition of the oligonucleotide probe and incubated at 25°C for 30 min. The double-stranded DNA oligodeoxynucleotide (ODN) for Egr1 binding that was identified as an Egr1 response element in the human p35 promoter was synthesized by the New Jersey Medical School Molecular Resource Facility. Its sequence is 5'-GCCGAGCGCCCCCGAGCGC-3'. We also mutated this site for binding and competition experiments and used 5'-GCCGAGCGCtttCaGCGC-3' as the mutated oligo.

Decoy Egr1 Oligodeoxynucleotide Transfection.
A double-stranded phosphorothioate Egr1 promoter decoy ODN was synthesized in the New Jersey Medical School Molecular Resource Facility. The Egr1 decoy ODN sequence is 5'-GCCGAGCGCCCCCGAGCGC-3' annealed to 5'-GCGCTCGGGGGCGCTCGGC-3'; and the sequence of the mutant Egr1 ODN is 5'-GCCGAGCGCTTTCAAGCGC-3' annealed to 5'-GCGCTTGAAAGCGCTCGGC-3'. For transient transfections, HL60 cells (1 x 106) were seeded in a six-well plate with fresh RPMI 1640 24 h before the transfection. Lipofectamine 2000 (Invitrogen, Carlsbad, CA) reagent was used for the transfection, according to the manufacturer’s protocol. The concentration of each ODN used for transfection was 250 nM. Fresh medium was added 24 h after addition of the ODN, the serum concentration was adjusted to 10%, and the cells were treated with 10 nM 1,25D3 for another 48 h.

Cell Differentiation Markers and Cell Cycle Analysis.
The CD14 and CD11b cell surface markers that are expressed when HL60 cells are induced to differentiate by 1,25D3 were determined by flow cytometry as described previously (26 , 27) . Cell cycle analysis was performed by propidium iodide staining of HL60 cells followed by a flow cytometric analysis, as described previously (4) .

Statistical Methods.
All of the experiments were repeated at least three times. Data are expressed as the mean ± SD. Analysis of data was by Student’s t test, taking P ≤ 0.05 as significant. The values were obtained using an IBM-compatible personal computer and Microsoft EXCEL program (SAS Institute, Cary, NC).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of Egr1 and p35 Parallels 1,25D3-Induced Differentiation of HL60 Cells in a 1,25D3 Dose-Dependent Manner.
It has been reported that Egr1 regulates p35 gene transcription in rodent neurons (13) . To initiate the testing of a possibility that Egr1 is also linked to the regulation of p35 gene expression in human leukemia HL60 cells induced to differentiate by 1,25D3, we determined protein levels of p35 and Egr1 after the exposure of HL60 cells to varying concentrations of the inducer. As shown in Fig. 1, A and BCitation , 1,25D3 induced p35 and Egr1 protein expression in a dose-dependent fashion in parallel with the induction of two markers of monocytic phenotype, CD14 and CD11b (Table 1)Citation . This is compatible with the hypothesis that both of these regulatory proteins participate in the induction of differentiation by 1,25D3.



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  		Fig. 1. Protein levels of Egr1 and p35Nck5a (p35) increase in parallel in a 1,25-dihydroxyvitamin D3 (1,25D3) dose-dependent manner, but the increase in Egr1 expression precedes the increase in p35 expression. A, protein levels of Egr1 and p35 after 48 h or 96 h exposure of HL60 cells to 1,25D3 at the indicated concentrations. Total cellular protein extracts (30 µg of each sample) were separated on a 7% or 12% SDS-PAGE gel, blotted onto nitrocellulose membrane, then probed with the indicated antibodies. Protein levels of calreticulin, a loading control, were determined by stripping the membrane and reblotting. B, quantitation of the absorbance (OD) of four immunoblots, one of which is illustrated in A. The mean of the ratio of the signals for either p35 or Egr1 to calreticulin are shown; bars, ±SD. The * indicates that the mean value is significantly (P < 0.05) different from the untreated control. C, time course of the expression of Egr1 and p35 mRNA as determined by reverse transcription-PCR (RT-PCR). HL60 cells were untreated or treated with 1,25D3 (10–8 M) from 12 h to 72 h. Total RNA was purified by the TRIzol reagent, and 2 µg of RNA from each sample was subjected to RT-PCR using 35 cycles for amplification of both Egr1 and p35. Note that the amounts of Egr1 mRNA increased in HL60 cells as early as 12 h after 1,25D3 treatment, whereas the increase in p35 mRNA was apparent only after 48 h of 1,25D3 treatment; C = control; D = 1,25D3-treated. D, quantitation of the absorbance of three RT-PCR analyses. Bars, ±SD.

 

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  		Table 1 Differentiation markers after treatment of HL60 cells with 1,25D3 for the stated timesa

 
We then determined by reverse transcription-PCR (RT-PCR) the effect of 1,25D3 on the kinetics of transcription of Egr1 and p35 genes. As shown in Fig. 1, C and DCitation , 1,25D3 up-regulated Egr1 transcription as early as 12 h, although p35 transcripts were not detected until 48 h after exposure to 1,25D3. These data are consistent with the possibility that p35 expression is regulated by Egr1 in HL60 cells but that either a threshold expression of Egr1 must be reached or that some other regulatory factors contribute to the increased expression of p35 in HL60 cells induced to differentiate by 1,25D3.

The 1,25D3 Increases the Binding of Egr1 to p35 Gene Promoter.
To investigate additionally the link between Egr1 and the expression of the p35 gene in HL60 cells responding to 1,25D3, we identified by sequence search of the gene bank database (accession no. AC002119) a putative Egr1-binding cis-acting element located 5' upstream of the human p35 gene coding sequence in the p35 promoter, positioned –557/–539 from the transcription start site (Fig. 2A)Citation . A similar Egr1-binding element was found to drive p35 expression in murine neuronal cells (13) . To test whether this element in the human p35 promoter could mediate Egr1-dependent up-regulation of p35, its binding to nuclear proteins extracted from 1,25D3-treated HL60 cells was determined by the electrophoretic mobility shift assay, as detailed in "Materials and Methods." As shown in Fig. 2BCitation , exposure of HL60 cells to 1,25D3 led to a marked increase in protein binding to this oligonucleotide (compare Lane 1 with Lane 2). The binding could be eliminated completely by adding a 10- or 50-fold excess of unlabeled Egr1 element oligonucleotide to the reaction mixture (Lanes 3 and 4) but not by a 50-fold excess of unlabeled Egr1 oligonucleotide mutated in four sites (Lane 8). Also, a 32P-labeled Egr1 mutant oligonucleotide did not show any Egr1 protein binding in the electrophoretic mobility shift assay (Lane 7). When nuclear extracts from 1,25D3-induced cells were incubated with antibodies against either Egr1 or vitamin D receptor (an irrelevant antibody control) before the performance of electrophoretic mobility shift assay, the antibody against Egr1 but not against vitamin D receptor was found to inhibit the binding to the Egr1 DNA element (Lanes 5 and 6). This experiment confirmed that the electrophoretic mobility shift band contained the Egr1 protein and that Egr1 protein binding to the p35 promoter increased after treatment of HL60 cells with 1,25D3.



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  		Fig. 2. The 1,25-dihydroxyvitamin D3 potentiates Egr1 binding to its response element located in the p35 promoter. A, schematic representation of the p35 gene 5' upstream of the transcription initiation start site (indicated as +1). B, gel shift analysis of nuclear protein binding to the Egr1 DNA element in p35 promoter before and after treatment of HL60 cells with 10–7 M 1,25-dihydroxyvitamin D3 for 48 h. Competition of binding was performed with the indicated excess of the unlabeled oligonucleotide used for the binding (Lanes 3 and 4) or with a mutant oligonucleotide, in which four residues in the Egr1 binding site (shown in Fig. 2ACitation ) were mutated (mutant oligo; Lane 8). An Egr1 antibody (Lane 5) partially blocked the binding, but an irrelevant antibody (antivitamin D receptor) did not (Lane 6). The mutant oligo also did not bind Egr1 (Lane 7) nor did it compete nuclear protein binding to the wild-type Egr1 oligo (Lane 8). ATG, start of the coding sequence. 1,25D3,1,25-dihydroxyvitamin D3; M, mutant oligo.

 
An Inhibitory Effect on p35 Gene Transcription and 1,25D3-Induced HL60 Cells Differentiation of a "Decoy" Oligonucleotide That Mimics a Sequence in the Egr1 Promoter.
To delineate additionally the involvement of Egr1 in 1,25 D3-induced up-regulation of p35 gene transcription, we performed promoter decoy experiments in which a large excess of an exogenous DNA element is introduced into the cells to compete with an endogenous transcription factor-binding element in a gene promoter (32 , 33) . In this case, HL60 cells were transfected with a wild-type or a mutated double-stranded oligonucleotide harboring the –557/–539 Egr1-cis-acting element present in the human p35 promoter and then treated the cells with 10 nM 1,25D3 for 48 h. To provide control groups, untransfected HL60 cells were treated without or with 1,25D3 for 48 h under the same conditions. Total cell RNA was extracted for purification and a subsequent RT-PCR analysis for Egr1 and CD14 gene transcripts. CD14 is a monocytic cell marker, the expression of which is induced during 1,25D3-induced HL60 cell differentiation. The data presented in Fig. 3Citation demonstrate that 1,25D3 induced an up-regulation of the expression of p35 gene and the monocyte-specific CD14 gene, but the up-regulation was decreased significantly in cells transfected with the wild-type decoy Egr1 oligonucleotide, compared with the mutant oligonucleotide-transfected or no oligo-transfected cells, although the mutant oligo displayed some nonspecific "oligo exposure" effect. Similarly, the proportion of cells positive for 1,25D3-induced monocyte-specific esterase reaction was reduced significantly in wild-type decoy Egr1 oligonucleotide-transfected cells (data not shown). These data suggest strongly that Egr1 at least contributes to the 1,25D3-induced expression of p35 and monocytic differentiation.



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  		Fig. 3. Egr1 decoy DNA oligo reduces the parallel expression of p35 and CD14 mRNA in HL60 cells treated with 1,25-dihydroxyvitamin D3. A, cells were untreated or treated with 1,25-dihydroxyvitamin D3 (10 nM) for 48 h in the presence or absence of a wild-type or mutated Egr1 decoy DNA oligo. B, two µg of total RNA from each sample were subjected to reverse transcription-PCR using 35 cycles (p35; CD14) or 25 cycles (ß-actin) for amplification. C, the relative amounts of either p35 or CD14 to the ß-actin mRNA. The values shown are mean from three experiments; bars, ±SD. The * indicate that the values in Lane 3 are significantly (P < 0.05) different from the values in Lane 4. The CD14 expression indicates monocytic differentiation. 1,25D3,1,25-dihydroxyvitamin D3.

 
Cdk5/p35 Kinase Inhibits the Activity of Erk MAPK Pathway by Phosphorylation of Raf1-Activated MEK1 in Terminally Differentiating HL60 Cells.
Recently, Sharma et al. (12) provided evidence that the Cdk5/p35 kinase complex regulates the Erk MAPK pathway by phosphorylating MEK1 on Thr286 in rat PC12 cells stimulated with nerve growth factor and in rat cortical neurons. Interestingly, the inhibitory phosphorylation occurs only if MEK1 is first activated by Raf1. This suggests that MEK1 is under sequential opposing controls by Raf1 and the Cdk5/p35 complex (i.e., MEK1 is activated through phosphorylation by Raf1 on Ser218 and Ser222 in the activation loop) and then can be inactivated through Thr286 phosphorylation in the proline rich domain by Cdk5/p35. This may be relevant to the previous work in our laboratory (4) , which showed that although the MEK/Erk MAPK pathway contributes to the early stage (up to 24–48 h) of 1,25D3-induced differentiation of HL60 cells, MAPK activity is reduced when the cells enter the terminal stage of differentiation. To address this point, we first determined the extent of 1,25D3-induced endogenous phosphorylation of Raf1 and of the in vitro phosphorylation of its downstream target MEK1 at an early point of differentiation. As shown in Fig. 4ACitation , immunoprecipitates of HL60 cell lysates with an antibody against Raf1 show an increase in P-Raf1, as well as in total Raf1, within 6 h of 1,25D3 exposure (top panels). When these immunoprecipitates were incubated with a GST-MEK1 fusion protein and 100 µM ATP under kinase reaction conditions (described previously in "Materials and Methods"), GST-MEK1 was found to be phosphorylated by Raf1 (Fig. 4ACitation , II, bottom panels). The phosphorylation of MEK increased when Raf1 kinase was immunoprecipitated from cell lysates obtained from cells treated with 1,25D3 (Fig. 4ACitation , II, bottom panels), showing that phosphorylation of Raf1 after exposure of the cells to 1,25D3 (Fig. 4ACitation , I, top panels) activated the kinase activity of Raf1.



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  		Fig. 4. Mitogen-activated protein/extracellular signal-regulated kinase kinase 1 (MEK1) can be phosphorylated sequentially in vitro by an active Raf1 then by the Cdk5 kinase complex. A, (I) 1,25-dihydroxyvitamin D3 (1,25D3) increases the abundance of the phosphorylated as well as total Raf1 protein in HL60 cells. Total protein lysates (200 µg) obtained from HL60 untreated control cells or from 1,25D3-treated cells, as indicated in the figure, were immunoprecipitated with an anti-Raf1 antibody or preimmune IgG as a negative control, electrophoresed on a SDS-PAGE gel, blotted onto nitrocellulose, then probed with the specific antiphosphorylated Raf1 antibody or anti-Raf1 antibody. (II) Raf1-dependent phosphorylation of mitogen-activated protein extracellular signal-regulated kinase kinase, using glutathione S-transferase-MEK1 as the substrate for an in vitro kinase assay and the immunoprecipitated complexes described in I. B, Cdk5/p35 phosphorylates Raf1-activated MEK1 in vitro. An autoradiogram of SDS-PAGE gel containing GST-MEK1 without preincubation (top panel) or after incubation with Raf1 immunoprecipitated from cells treated for 6 h with 1,25D3 (middle panel). The Raf1-activated MEK was then incubated with Cdk5 immunoprecipitated complex from untreated HL60 cells (Lane 1), or from HL60 cells treated with 1,25D3 (10 nM) for 48 h (Lane 2), or with preimmune IgG immunoprecipitated complex (Lane 3), and then subjected to SDS-PAGE and autoradiography. Cdk5 content was detected after the kinase reaction by transferring the immunoprecipitated Cdk5 complex from the SDS-PAGE gel to the membrane and blotting with an antibody against Cdk5 (bottom panel). Note the high level of MEK1 phosphorylation after its preincubation with activated Raf1 (Lane 2, middle panel), but not without the preincubation (top panel), and a weak signal after an incubation with Cdk5 complex from control cells, which do not express p35 (Lane 1, middle panel). MEK, mitogen-activated protein extracellular signal-regulated kinase kinase; GST, glutathione S-transferase; IP, immunoprecipitation; IB, immunoblotting.

 
Then we tested whether the Cdk5/p35 kinase complex obtained from HL60 cells can phosphorylate recombinant MEK1 in vitro. In this experiment, HL60 cells were incubated without or with 10 nM 1,25D3 for 48 h, cell extracts immunoprecipitated with an antibody against Cdk5, and the immunoprecipitated complex was used for kinase reaction using GST-MEK1 as a substrate. As shown in Fig. 4BCitation (top panel), this immunoprecipitated complex does not phosphorylate GST-MEK1 not prephosphorylated by Raf1 (GST-MEK1-inactive). However, if GST-MEK1 is first phosphorylated by Raf1 immunoprecipitated from 1,25D3-treated cells, as in the experiment shown in Fig. 4ACitation (II, Lane 2), it was phosphorylated additionally by the Cdk5/p35 complex, as shown by the incorporation of radioactive phosphorus (Fig. 4BCitation , middle panel, Lane 2). It can be noted also that Cdk5 immunoprecipitated from HL60 cells not exposed to 1,25D3, in which p35 is not expressed (Fig. 1, A and C)Citation , did not phosphorylate active MEK1 (compare signal in Lane 1 with the background signal shown in Lane 3, middle panel in Fig. 4BCitation ). The presence of Cdk5 protein in the immunoprecipitates was confirmed by immunoblotting (Fig. 4BCitation , bottom panel). Thus, Cdk5 can phosphorylate MEK1 activated by Raf1 from 1,25D3-treated HL60 cells.

To determine whether Cdk5/p35 phosphorylation of Raf1-activated MEK1 alters MEK1 kinase activity, we tested its ability to phosphorylate its principal downstream target in HL60 cells, Erk2. First, as in the experiment presented in Fig. 4ACitation , HL60 cells were incubated without or with 1,25 D3 for 6 h, the cell extracts immunoprecipitated with an antibody against Raf1, and the immunoprecipitated complex used to phosphorylate GST-MEK1. We designated the GST-MEK1 phosphorylated by Raf1 from control cells "GST-MEK1-Raf-activated1" (as in Fig. 5Citation , Lanes 3 and 4) and from the 1,25D3-treated cells as "GST-MEK1-Raf-activated2" (Fig. 5Citation , Lanes 5 and 6). Then, as in the experiment shown in Fig. 4BCitation , HL60 cells were treated without or with 10 nM 1,25D3 for 48 h, the cell extracts immunoprecipitated with an antibody against Cdk5, and the immunoprecipitated complex was used for the kinase reactions in vitro using the following as sequential substrates: (a) GST-MEK1 ("inactive") and GST-Erk2; (b) GST-MEK1-Raf-activated1 and GST-Erk2; or (c) GST-MEK1-Raf-activated2 and GST-Erk2. As shown in Fig. 5Citation , Erk2 was not phosphorylated by an incubation with GST-MEK1 (inactive) together with the Cdk5 complex immunoprecipitated from untreated cells (Lane 1) or from 1,25D3-treated cells (Lane 2). However, Erk2 was phosphorylated by GST-MEK1-Raf-activated1 incubated with the Cdk5 complex obtained from untreated HL60 cells (Fig. 5Citation , Lane 3), and the Erk2 phosphorylation was more marked when GST-MEK1-Raf-activated2 was used (Lane 5). In contrast, Erk2 phosphorylation by MEK1 was completely inhibited by an incubation of GST-MEK, Raf-activated1, or activated2, with the Cdk5/p35 complex obtained from cells treated with 1,25D3 for 48 h (Lanes 4 and 6). These experiments suggest strongly that the Cdk5/p35 complex can phosphorylate MEK1 only when MEK1 is first phosphorylated by Raf1 and that phosphorylation of MEK1 by the Cdk5 complex results in the inhibition of the ability of MEK1 to phosphorylate Erk2.



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  		Fig. 5. Phosphorylation of recombinant glutathione S-transferase (GST)-extracellular signal-regulated kinase 2 (Erk2) by activated mitogen-activated protein/extracellular signal-regulated kinase kinase 1 (MEK1) is inhibited by Cdk5 immunoprecipitated from 1,25-dihydroxyvitamin D3 (1,25D3)-treated HL60 cells. A, the outline of the protocol for a two-stage experiment. The assignment of serine residues in MEK1 that are phosphorylated by Raf1 is based on a published report (12) . B, phosphorylation of an activated MEK1 by immunoprecipitated Cdk5 complexes results in the inhibition of its ability to phosphorylate Erk2. In stage 1, GST-MEK1 was incubated without Raf1 (Lanes 1, 2, and 7) or incubated in vitro with Raf1 immunoprecipitated from lysates of HL60 cells untreated (Lanes 3 and 4) or treated with 1,25D3 for 6 h (Lanes 5 and 6). In stage 2, the inactive mitogen-activated protein extracellular signal-regulated kinase kinase (MEK; Lanes 1 and 2) or Raf-activated MEK (Lanes 3–6) was incubated with the GST-Erk2 substrate in the presence of Cdk5 complexes immunoprecipitated from lysates of cells untreated by 1,25D3 (Lanes 1, 3, and 5) or 1,25D3-treated cells (Lanes 2, 4, and 6), which express p35 (as shown in Figs. 1Citation and 6Citation ). Note that phosphorylation of Erk2 was not detected when MEK was not preincubated with Raf1 (Lanes 1, 2, and 7). However, Erk2 was phosphorylated by MEK preincubated with Raf1 from control cells (Lane 3), and the phosphorylation increased if Raf1 was immunoprecipitated from lysates of cells treated with 1,25D3 for 6 h (Lane 5), which increases P-Raf1 levels (Fig. 4, AI)Citation . Importantly, although the coincubation with Cdk5 complexes immunoprecipitated from control HL60 cells did not interfere with the phosphorylation of Erk2 by MEK (Lanes 3 and 5), coincubation with Cdk5 complexes obtained from cells treated with 1,25D3 for 48 h inhibited the phosphorylation of Erk2 (Lanes 4 and 6). Lane 7 is a negative control with preimmune IgG immunoprecipitate in place of Cdk5 immunoprecipitate from HL60 cells. The immunoglobulin bands (IgG H and IgG L) are shown to demonstrate their resolution from Erk2 and to indicate essentially equal loading of the lanes. The bottom panel shows that the input GST-Erk2 is constant. IP, immunoprecipitation; IB, immunoblotting.

 
Up-Regulation of p35 Expression Coincides with the Down-Regulation of Erk Activation and a G1 Block.
To explore whether it is feasible that the Cdk5/p35 kinase complex can down-regulate the activation of Erk1/2 in intact HL60 cells, we treated HL60 cells with 10 nM 1,25D3 for 6–96 h and determined the expression of p35 and phosphorylation of Erk1/2 by immunoblotting. As shown in Fig. 6Citation , P-Erk1/2 levels decreased after 24 h, the time at which p35 expression was increasing, consistent with the data presented above, which showed the inhibition of Erk2 phosphorylation by activated MEK1 (Fig. 5)Citation . Importantly, the kinetics of the increase in p35 expression coincided with the onset of the accumulation of the cells in the G1/G0 compartment (Fig. 6)Citation , a feature of terminal differentiation. Although there is also an accumulation of p27Kip1 and cyclin D1 with kinetics similar to the cell cycle block observed here (34 , 35 ; data not shown), this suggests that the arrest occurs in late G1 phase, as proposed by Blagosklonny (36) for a late G1"hypermitogenic arrest." Thus, p35 appears to contribute to the cell cycle arrest of differentiating cells, probably by an inactivating phosphorylation of MEK1 by the Cdk5/p35 complex.



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  		Fig. 6. Down-regulation of extracellular signal-regulated kinase (Erk)1/2 by a prolonged exposure to 1,25-dihydroxyvitamin D3 (1,25D3) coincides with the increased expression of p35 and the onset of the G1 to S-phase block. A, an immunoblot analysis of the levels of phospho-Erk1/2, total-Erk1/2, and p35 proteins after an exposure of HL60 cells to 1,25D3 (10–8 M) for the indicated times. The double arrows show Erk1 higher band, Erk2 lower band, and their phosphorylated forms. B, the absorbance ratios of phospho-Erk to total Erk. C, the ratios of p35 to the nonspecific band plotted versus time in the presence of 1,25D3. Note the peak increase in phospho-Erk1/2 at 24 h and the increase in p35 protein levels after 48 h of 1,25D3 treatment. D, cell cycle analysis by flow cytometry after propidium iodide staining. The ratio of G1 to S-phase cells increased after 48-h treatment of HL60 cells with 1,25D3 in parallel with the increased expression of p35 shown in C, whereas there was no progressive G2-M arrest. The actual mean percentage members at each time point after addition of 1,25D3 were as follows: control, G1:45.5, S:45.7, G2:8.8, 6 h, G1:46.9, S:44.9, G2:8.2; 12 h, G1:46.6, S:41.1, G2:12.3; 24 h, G1:51, S:40.3, G2:8.7; 48 h, G1:56.9, 5:30.3, G2:12.8; 96 h, G1:79.1, S:10.1, G2:10.8. NS, nonspecific; bars, ±SD.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The realization that the expression of p35 is not strictly limited to neuronal cells (22, 23, 24, 25, 26, 27, 28, 29) opened new lines of investigation into the mechanistic basis of terminal differentiation. On the basis of the postulated existence of a common neurohematopoietic stem cell (37 , 38) , we conducted several studies that establish that Cdk5, activated by the so-called "p35 neuron-specific Cdk5 activator, p35Nck5a," plays a role in monocytic differentiation, both in normal human hematopoiesis and in human leukemia cells induced to differentiate to the monocytic phenotype by 1,25D3 (26, 27, 28, 29) . An active Cdk5 was also found in rat promyelocytic leukemia cells, suggesting that it may characterize myeloid hematopoietic cells of species other than human (39) . We have now examined a plausible signaling pathway that is triggered in human leukemia HL60 cells by 1,25D3-induced up-regulation of Egr1, shown here to at least contribute to the expression of p35. This cyclin-like protein then activates Cdk5 to phosphorylate and, thus, disable active MEK1, a key component of the mitogen-activated Ras/Raf/MEK/Erk pathway. As a result, the Erk MAPK pathway is interrupted, and one consequence is that 1,25D3-treated cells reduce their proliferation rate (40) and accumulate in the G1/G0 cell compartment, aided by the increased levels of the p27Kip1, an inhibitor of Cdks active in the G1 phase of the cell cycle (34) . This sequence of events is presented schematically in Fig. 7Citation .



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  		Fig. 7. A schematic representation of the findings presented in this paper, their interpretation, and data from previous reports. Proliferation of normally growing progenitor-like HL60 cells is driven by mitogens, other growth factors, and cytokines (pathway shown by open arrows). Kinase suppressor of ras is up-regulated by 1,25-dihydroxyvitamin D3 and amplifies the differentiation signal in HL60 cells (6) . When HL60 cells are treated with 1,25-dihydroxyvitamin D3, the Egr1 gene is up-regulated. The Egr1 protein subsequently activates p35 gene transcription by binding to its promoter region. Cdk5 and p35 then form a complex and phosphorylate mitogen-activated protein/extracellular signal-regulated kinase kinase 1 on Thr286 as shown in murine cells (12) and, thus, inactivate its ability to phosphorylate its downstream target Erk. This inhibitory phosphorylation occurs only if mitogen-activated protein/extracellular signal-regulated kinase kinase 1 is first phosphorylated and activated by Raf1 on Ser218 and Ser222. We suggest that as the result of Cdk5/p35 activity, the extracellular signal-regulated kinase mitogen-activated protein kinase pathway is shut off, and this contributes to the p27Kip1 up-regulation (37) , a late G1 hypermitogenic (35) block, inhibition of cell proliferation (40) , and the onset of terminal differentiation of the myeloid cells (pathway shown by shaded arrows). KSR, kinase suppressor of ras; 1,25D3, 1,25-dihydroxyvitamin D3; MEK1, mitogen-activated protein/extracellular signal-regulated kinase kinase 1; Erk, extracellular signal-regulated kinase.

 
It has been known for some time that the zinc finger transcription factor Egr1 is expressed selectively during monocyte/macrophage differentiation and is indeed essential for this form of differentiation (41 , 42) . Consistent with these reports, it was shown recently in this laboratory that treatment of HL60 cells with 1,25D3 up-regulates Egr1 expression (40) . We have now identified a putative Egr1 binding site in the promoter of the human p35 gene (Fig. 2A)Citation and show that Egr1 can bind to this element (Fig. 2B)Citation , whereas a large excess of oligonucleotides containing this element (promoter decoy) has a dominant-negative effect on the expression of p35 (Fig. 3)Citation . This suggests strongly that Egr1 contributes to the regulation of p35 expression, although the contribution of other transcription factors, such as Sp1 (43) , cannot be excluded. Indeed, the GC boxes that can bind both Egr1 and Sp1 have been reported to act in concert (44) . The contribution of the members of the Sp1 family to the up-regulation of p35 in this system remains to be investigated, but because there are several members of this family, this will be a major task.

In the p35 promoter decoy experiment, we observed that in addition to the inhibition of p35 expression, markers of monocytic differentiation CD14 and monocyte-specific esterase were also reduced (Fig. 3Citation ; data not shown). A possible explanation is that Cdk5, activated by p35, targets a regulator of monocytic differentiation. Although there could be many such targets, we were able to show that active Cdk5 can inhibit the catalytic activity of MEK1, but only if it was phosphorylated previously and, thus, activated by Raf1 (Figs. 4Citation and 5Citation ), similar to the situation in rodent neuronal cells (12) . In these cells, the activating phosphorylation by Raf1 takes place on serines 218 and 222, whereas the inactivating phosphorylation by Cdk5 takes place on threonine 286 of MEK1 (12) , suggesting that the different conformational changes in the protein induced by these phosphorylations restrict possible contacts with target proteins in different ways. These amino acids are conserved in human MEK1. Thus, cross-talk between p35 and MEK-Erk mitogen-activated protein pathway may regulate proliferation of differentiating myeloid cells.

The onset of terminal differentiation induced by 1,25D3 is recognized by the accumulation of cells in the G1/G0 compartment, which is attributed to the increased levels of p27Kip1 and perhaps p21Cip1, inhibitors of the G1 Cdk/cyclin complexes (34 , 45) . Mitogens have been reported to be inhibitory to p27Kip1 up-regulation (46) so the inhibition of the Erk MAPK pathway by Cdk5/p35 may be a factor in allowing an accumulation of p27Kip1 and the consequent onset of the "hypermitogenic" G1 arrest. This may in turn facilitate differentiation, because differentiation occurs predominantly in the G1 phase of cell cycle (47) . Future studies should elucidate the relationship of p27Kip1 up-regulation to the increased Cdk5/p35 activity and clarify additionally details of the mechanisms of the control of Cdk5 and p35 expression.

In the present report, we provide evidence for a novel pathway that signals events of terminal monocytic differentiation through Egr1 and the Cdk5/p35 complex. This finding may have important clinical implications, as pharmacological inhibitors of Cdk5 are already undergoing Phase I and II clinical trials (48) .


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Milan Uskokovic (BioXell, Nutley, NJ) for a generous gift of 1,25-dihydroxyvitamin D3, Dr. Le Hua Tsai (Harvard Medical School, Boston, MA) for a helpful discussion, Drs. Michael Danilenko (Ben Gurion University of the Negev, Beer-Sheva, Israel), and Robert Murray (University of Toronto, Toronto, Ontario, Canada) for comments on the manuscript, and Terri McNeil for expert secretarial assistance.


   FOOTNOTES
 
Grant support: NIH Grant RO1-CA44722-14 from the National Cancer Institute, and the United States-Israel Binational Science Foundation Grant 2001041 (Q. Wang).

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.

Note: F. Chen and Q. Wang contributed equally to this work. Present address for F. Chen is Department of Surgery, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103.

Requests for reprints: George P. Studzinski, Department of Pathology and Laboratory Medicine, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103. Phone: (973) 972-5869; Fax: (973) 972-7293; E-mail: studzins{at}umdnj.edu

Received 3/ 4/04. Revised 4/16/04. Accepted 5/ 5/04.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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