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[Cancer Research 64, 370-377, January 1, 2004]
© 2004 American Association for Cancer Research

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Retinoblastoma Protein and CCAAT/Enhancer-Binding Protein ß Are Required for 1,25-Dihydroxyvitamin D3-Induced Monocytic Differentiation of HL60 Cells

Yan Ji and George P. Studzinski

Department of Pathology and Laboratory Medicine, The 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
 
Derivatives of vitamin D (deltanoids) are well known to have the ability to induce differentiation of a variety of malignant cells, including human leukemia cells, but the signaling pathways that lead to such an outcome are unclear. In this study we investigated the role of the retinoblastoma protein (pRb) and the CCAAT/enhancer-binding protein (C/EBP) ß in 1,25-dihydroxyvitamin D3 (1,25D3)-induced monocytic differentiation of human leukemia HL60 cells. It was found that in this system, pRb is up-regulated within 12 h of exposure to the inducer, and the kinetics of its increase parallel the appearance of the early markers of differentiation, CD14 and monocyte-specific esterase. The increase in pRb expression was accompanied by a similar increase in C/EBPß protein, and these two proteins coimmunoprecipitated, suggesting formation of a complex. Oligonucleotides antisense to pRb or C/EBPß (but not to C/EBP{alpha}) or containing the C/EBP-binding sequence ("decoys"), all inhibited 1,25D3-induced differentiation. Inhibition of signaling by vitamin D receptor or by mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase and c-Jun-NH2-terminal kinase pathways using pharmacological inhibitors ZK159222, PD98059, or SP600125, respectively, inhibited pRb and C/EBPß expression and differentiation in a coordinate manner. In contrast, inhibition of the p38MAPK pathway by SB202190 potentiated differentiation and the up-regulation of pRb and C/EBPß. We suggest that 1,25D3 may signal monocytic differentiation of HL60 cells in a vitamin D receptor-dependent manner that includes activation of extracellular signal-regulated kinase and c-Jun-NH2-terminal kinase MAPK pathways, which then up-regulate pRb and C/EBPß expression and in turn initiate the differentiation process.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Differentiation therapy is conceptually an elegant approach to the eradication of neoplastic cells from the human body because cytotoxicity is avoided, whereas normal mature cells are unaffected by the differentiation agents. Clear-cut successes have already been achieved, such as the use of retinoids for the treatment of acute promyelocytic leukemia (1, 2, 3) . Derivatives of vitamin D, dubbed "deltanoids" in analogy to the retinoids (4) , also hold promise for use as components of differentiation regimens, and several are currently used in clinical trials, e.g., EB 1089 in patients with advanced breast or colorectal cancer (5) , and this deltanoid has already been approved for the treatment of advanced hepatocarcinoma (6) . However, progress in generation of even more effective deltanoids should be accelerated by a better understanding of the mechanisms by which these compounds signal differentiation.

The retinoblastoma protein (pRb), a product of a prominent cancer gene (7, 8, 9, 10) , has a less well appreciated or understood role in normal cell differentiation. It is a ubiquitously expressed protein that exists at several levels of phosphorylation (11 , 12) . When hypophosphorylated, pRb can block the cell cycle traverse by binding members of the E2F transcription factor family through its pocket domain (13 , 14) , thus preventing E2F proteins from transactivating the genes that encode various components of the machinery required for the cell cycle traverse and DNA replication (15, 16, 17) . However, pRb can also interact with transcription factors that regulate differentiation, such as MyoD (18) , HBP1 (19 , 20) , CCAAT/enhancer-binding protein (C/EBP) {alpha} (21) , and C/EBPß (22) . Thus, pRb is implicated in myogenesis (18 , 23) , adipogenesis (24 , 25) , osteogenesis (26) , and hematopoiesis (27) . Notably, pRb binding to C/EBPß is associated with 12-O-Tetradecanoylphorbol-13-acetate-induced macrophage differentiation of myelomonocytic leukemia U937 cells (22) . This is in keeping with the observation that in normal hematopoiesis, C/EBPß is expressed in the monocyte lineage, whereas C/EBP{alpha} and C/EBP{delta} are predominantly expressed in the granulocyte and eosinophil lineages, respectively (21 , 28 , 29) .

What controls pRb and C/EBP expression in hematopoietic cells is not established. In the case of deltanoid-induced differentiation, it was demonstrated that there is an increased expression of pRb before the onset of the G1 block (30) and that it is accompanied by an increased activity of three mitogen-activated protein kinase (MAPK) pathways (30, 31, 32, 33) , but the relationship between these events is not clear. We have addressed this issue and further investigated the kinetics of retinoblastoma (RB) gene up-regulation in 1,25-dihydroxyvitamin D3 (1,25D3)-treated HL60 cells, as well as the participation of C/EBPß in this form of differentiation. Data presented here show that both pRb and C/EBPß are required for 1,25D3-induced differentiation of HL60 cells, form a complex, and appear to be under the control of vitamin D receptor (VDR) and MAPK-transduced signals.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Antibodies.
1,25D3 was a gift from Dr. Milan Uskokovic (Bio Xell, Inc., Nutley, NJ). The antibodies used to detect pRb (IF-8, mouse polyclonal antibody), VDR (C-20), C/EBP{alpha} (14AA, rabbit polyclonal antibody), C/EBPß (C19, rabbit polyclonal antibody), Crk-L (C-20, rabbit polyclonal antibody), antimouse IgG-horseradish peroxidase, and antirabbit IgG-horseradish peroxidase were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mitogen-activated protein/ERK kinase/extracellular signal-regulated kinase inhibitor PD98059 was purchased from Cell Signaling Technology (Beverly, MA). The p38 kinase inhibitor SB202190 was purchased from Calbiochem-Novabiochem Corp. (San Diego, CA). c-Jun-NH2-terminal kinase inhibitor SP600125 was a generous gift from Signal Research Division, Celgene Corp. (Warren, NJ), and ZK159222 was a gift from Schering AG (Berlin, Germany).

Cell Culture.
HL60-G cells (34) , a subclone of human promyelocytic leukemia cells (35) , were grown in suspension culture at 37°C in a closed atmosphere in RPMI 1640 (Mediatech, Washington, D.C.) with 10% heat-inactivated, defined iron-supplemented bovine calf serum (Hyclone, Logan, UT) and 1% L-glutamine. The cell number and viability were determined by hemocytometer counts and trypan blue (0.4%) exclusion. For all experiments, the cells were suspended at 2.5 x 105 cells/ml fresh medium containing the desired concentrations of 1,25D3. Cells in the control groups received an equal volume of ethanol in which 1,25D3 was dissolved. Each experiment was repeated at least three times.

Determination of Markers of Differentiation.
These methods were described previously (36) . Briefly, aliquots of 1 x 106 cells were washed twice with 1x PBS and then incubated with 0.5 µl of MY4-RD1 and 0.5 µl of -FITC (Coulter, Miami, FL) at room temperature for 45 min to analyze the expression of surface cell markers CD14 and CD11b, respectively. The cells were then suspended in 0.5 ml of 1x PBS and analyzed using an Epics Profile II instrument (Coulter Electronics, Hialeah, FL). The thresholds for side scatter and forward scatter were set using {gamma}-1 FITC/{gamma}-2A phycoerythrin as subclass control. CD11b FITC/CD14 PE-positive sample was used to adjust color compensation, and 104 cells were analyzed in each sample.

For assessment of differentiation by monocyte-specific esterase (MSE), also known as nonspecific esterase, smears were made by resuspending 2 x 106 cells in 100 µl of 1x PBS and spreading them on slides. The air-dried smears were fixed for 30 s in formalin (25%) acetone (45%) mixture buffer and then washed with distilled H2O and stained for 45 min at room temperature with the following solution: 67 mM sodium phosphate buffer (pH 7.6; 8.9 ml); hexazotized pararosaniline (0.6 ml); 10 mg of {alpha}-naphthyl acetate; and 0.5 ml of ethylene glycol monomethyl ether. The MSE-positive cells were determined by counting 500 cells in each group under a light microscope.

Western Blotting.
Western blotting was performed using nuclear extracts. Twenty µg of protein samples were separated on SDS-PAGE (7% for pRb and 10% for all of the other proteins) gels and transferred to nitrocellulose membranes (Amersham Pharmacia, Little Chalfont, United Kingdom). The membranes were blocked with 5% milk in Tris-buffered saline/0.1% Tween 20 for 1 h, subsequently blotted with primary antibodies, and then blotted with horseradish peroxidase-conjugated secondary antibody for 1 h. The protein bands were visualized with a chemiluminescence assay system (Amersham). The membranes were stripped according to the manufacturer’s protocol (Amersham) and reprobed with Crk-L. The absorbance of each band was quantitated using an Image QuaNT program (Molecular Dynamics, Sunnyvale, CA).

Reverse Transcription-PCR.
Total RNA was extracted using Trizol reagent. GeneAmp RNA PCR Core Kits (Perkin-Elmer, Boston, MA) were used. The primers were as follows: (a) RB upstream primer (5'-TACCTAGCTCAAGGGTTAAT-3') and RB downstream primer (5'-TAGCCATATGCACATGAATG-3'); (b) C/EPBß upstream primer (5'-GTTCTTGACGTTCTTCGGCCG-3') and C/EPBß downstream primer (5'-TGGACAAGCACAGCGACGAGT-3'), and (c) ß-actin upstream primer (5'-TGACGGGGTCACCCACACTGTGCCCAGCTA-3') and ß-actin downstream primer (5'-CTAGAAGCATTTGCCGGTGGACGATGGAGGG-3'). Reverse transcription-PCR was performed according to the manufacturer’s recommended procedure. For reverse transcription, samples were incubated in a Perkin-Elmer GeneAmp PCR System 9600 at 42°C for 15 min and then incubated at 99°C for 5 min and 5°C for 5 min. For PCR, samples were incubated in GeneAmpPCR System 9600 as follows: 95°C for 105 s; 35 cycles of 95°C for 15 s and 60°C for 30 s; and 72°C for 7 min. The reverse transcription-PCR products were separated in 1.2% agarose gels and stained with ethidium bromide. The intensities of the bands were measured using Image QuaNT program (Molecular Dynamics). For real-time reverse transcription-PCR, LighterCycler-FastStart DNA Master SYBR Green I kit (Roche, Indianapolis, IN) was used. Reverse transcription was performed as referred to above, and PCR was performed according to the manufacturer’s recommended procedure. Briefly, 2 µl of cDNA samples from reverse transcription reaction were mixed with 2 µl of SYBR Green I, 2.4 µl of MgCl2 (25 mM), 1 µl of upstream and downstream primers (7.5 µM) each, and 11.6 µl of PCR-grade distilled H2O in a capillary and placed in the LightCycler. The PCR reaction temperature was set as described above. Data were analyzed using Relative Quantification software from Roche.

Antisense (AS) Oligonucleotides.
AS oligonucleotides were used to inhibit the expression of products of the genes of interest. Phosphorothioate oligodeoxynucleotides were synthesized by the Molecular Resource Facility of the New Jersey Medical School. The sequences of the phosphorothioate-AS-RB oligonucleotide and C/EBP{alpha} and C/EBPß AS oligodeoxynucleotides that target the translation initiation sites of RB gene, C/EBP{alpha} gene, and C/EBPß gene, respectively, were as follows: AS-RB (5'-GGGGGTTTTGGGcGGCATGAC-3') and scrambled (SC)-RB (5'-GTGCGAGTGGCGTGAGTGCGT-3'); AS-C/EBP{alpha} (5'-GAAGTCGGCCGACTCCAT-3') and SC-C/EBP{alpha} (5'-ATGCATGCATGCAGCGCC-3'); and AS-C/EBPß (5'-CACCAGGCGTTGCATGAA-3') and SC-C/EBPß (5'-ACTGACTGACTGA-CGACG-3'). HL60-G cells were preincubated with the AS oligonucleotides at a final concentration of 5 µM in the culture medium for 24 h before treatment with 1,25D3.

Decoy Oligonucleotide Inhibition Assay.
A decoy oligonucleotide inhibition assay was used to study the role of various transcription factors (37) . Decoy and mutant decoy oligonucleotides were phosphorothioated oligonucleotides, synthesized by the Molecular Resource Facility of the New Jersey Medical School. The decoy C/EBP oligonucleotides sequences were as follows: 5'-tgcaGATTGCGCAATCtgca-3' and its complement; and SC C/EBP (5'-tgcaGAGACTAGTCTCtgca-3') and its complement. HL60-G cells were preincubated with the decoy at a final concentration of 5 µM of the oligonucleotides in culture medium for 24 h before treatment with 1,25D3.

Coimmunoprecipitation.
Seize Primary Mammalian Immunoprecipitation Kit (Pierce Biotechnology, Rockford, IL) was used. Immunoprecipitations were performed according to the manufacturer’s recommended procedures. First, 50 µg of anti-C/EBPß or anti-RB antibody were coupled to 100 µl of AminoLink Plus coupling gel in a spin column and incubated overnight at 4°C. Then, 200 µl of whole cell protein extract were incubated at 4°C with antibody coupling gel overnight. Next, immunoprecipitated proteins were eluted from the column and run on SDS-PAGE gel (7% for pRb and 10% for C/EBPß), transferred to a membrane, and probed with anti-pRb or anti-C/EBPß. The protein bands were visualized with a chemiluminescence assay system (Amersham Pharmacia Biotech. Inc).

Statistical Methods.
All experiments were repeated a minimum of three times. Significance of differences between mean values was assessed by ANOVA analysis followed by the Bonferroni post-test. All computations were performed using an IBM personal computer using Microsoft EXCEL + ANALYSE-IT Program.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The Kinetics of Up-Regulation of RB Gene Expression Parallel the Expression of Early Markers of Monocytic Differentiation.
It was observed previously that levels of the pRb are increased in HL60 cells treated with concentrations of 1,25D3 that are insufficient to cause cell cycle arrest but that induce markers of differentiation (30) . This showed that in addition to its well known role in controlling the cell cycle transitions (15 , 38) , pRb is likely to function in cellular events associated with monocytic differentiation, which precede the cell cycle blocks induced by 1,25D3. However, the relationship of pRb up-regulation to the early differentiation events of 1,25D3-treated cells was not evident from those studies. Therefore, to determine whether the up-regulation of pRb expression can be considered as one of the earliest detectable events during 1,25D3-induced differentiation, we compared the kinetics of this up-regulation and of the appearance of two early markers of monocytic differentiation, CD14 and MSE, also known as the nonspecific esterase. Interestingly, the increase in pRb mRNA closely paralleled the appearance of CD14 and MSE in HL60 cells induced to differentiate with either 1 or 10 nM 1,25D3 (Fig. 1, A and B)Citation . Furthermore, there was also a good correlation between increased levels of pRb and these early differentiation markers (Fig. 1C)Citation , whereas the myeloid marker CD11b, which appears later in monocytic differentiation, and the RB-related pocket protein p130, which is associated with replicative quiescence (39, 40, 41) , showed increases with kinetics that were different from those observed for CD14, MSE, and pRb (compare Fig. 1, C and DCitation ). Thus, the up-regulated expression of pRb is an early event in 1,25D3-induced monocytic differentiation of HL60 cells, whereas the p130 pocket protein and CD11b marker signify a more mature monocytic phenotype.



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  		Fig. 1. Kinetics of the up-regulation of retinoblastoma protein parallel the appearance of the early markers of differentiation. HL60-G cells were treated with 1 (A) or 10 nM (B) 1,25-dihydroxyvitamin D3. The differentiation marker CD14 was determined at the indicated intervals by flow cytometry, monocyte-specific esterase was determined by cytochemistry, and retinoblastoma mRNA was determined by quantitative real-time reverse transcription-PCR. The protein levels of retinoblastoma protein (C) and p130 (D) were determined by Western blots of extracts of cells treated with 5 nM 1,25-dihydroxyvitamin D3. Note the different time scale in A/B and C/D. Error bars represent the SDs in this and all subsequent figures. Fold increase refers to the levels of retinoblastoma or p130 mRNA.

 
The C/EBPß Is Up-Regulated in HL60 Cells by 1,25D3 in Concert with pRb and Forms a Complex with It.
Rb protein is known to function by binding to and modifying the activity of transcription factors, including members of the E2F and the C/EBP families (18 , 22 , 25) . E2F transcription factors regulate many genes whose products are required for the G1 traverse and DNA replication (15 , 17) , and their binding to pRb negatively influences their activity. In contrast, binding of pRb to members of the C/EBP transcription factor family has been suggested to have a positive effect on their functions, which include activation of genes required for differentiation of several cell types (24 , 28 , 29 , 42) . We therefore investigated in the 1,25D3-HL60 cell differentiation model to determine whether there is an interaction between pRb and C/EBPß, previously implicated in monocytic differentiation (22) . Immunoprecipitates of C/EBPß showed the presence of pRb, and in a reciprocal experiment, C/EBPß was detected (Fig. 2)Citation , suggesting formation of a complex, as detected previously in another cell line (22) . Additionally, we found that 1,25D3 increased the expression of C/EBPß in a dose-dependent manner, which paralleled the increased expression of pRb in these cells (Fig. 2)Citation . Quantitation of the signals on Western blots showed a similar magnitude of the increase, with the maximum being 5-fold higher pRb and 4-fold higher C/EBPß protein levels in cells treated with >=10 nM 1,25D3 (Fig. 2C)Citation . Thus, pRb and RB mRNA expression increased in a time- and dose-dependent manner after exposure to 1,25D3 and correlated with an increased expression of C/EBPß and early markers of differentiation



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  		Fig. 2. Dose-dependent up-regulation of retinoblastoma protein and CCAAT/enhancer-binding protein ß and formation of a complex. A, Western blots. Crk-L was determined as a loading and transfer control, and the signal was used for subsequent quantitation. B, Western blots after reciprocal immunoprecipitation. C, quantitation of three similar experiments illustrated in A. *, P < 0.05 relative to the control group.

 
Up-Regulation of RB Gene Expression Is Necessary for 1,25D3-Induced Differentiation of HL60 Cells.
We initially tested the hypothesis that pRb enhances the ability of C/EBPß to activate the transcription of differentiation-related genes by reducing the expression of pRb by an AS oligonucleotide (AS-RB), followed by a determination of the ability of 1,25D3 to induce differentiation. As illustrated in Fig. 3ACitation and quantitated in Fig. 3BCitation , the AS-RB oligonucleotide effectively blocked the expression of early markers of differentiation in cells exposed to a low concentration (1 nM) of 1,25D3 for 24 h, but an exposure to an oligonucleotide with a SC sequence (SC-RB) did not. Although quantitation of the expression of CD11b was also attempted (Fig. 3A)Citation , no significant expression of this late myeloid marker was noted at this time (24 h) of 1,25D3 exposure. A Western analysis of proteins extracted from these cells showed an almost complete inhibition by AS-RB, but not by SC-RB, of the 1,25D3-induced up-regulation of pRb (Fig. 3, C and D)Citation . This analysis also demonstrated that the expression of C/EBPß and VDR is not affected by AS-RB, indicating that the presence of up-regulated C/EBPß, or VDR, is not sufficient for the induction of differentiation (Fig. 3, C and D)Citation . Also, it is evident that the expression of C/EBPß and VDR is not regulated by pRb because they are unaffected by the depletion of pRb by the AS-RB.



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  		Fig. 3. Antisense retinoblastoma (RB) blocks differentiation induced by 1,25-dihydroxyvitamin D3 (1,25D3) in HL60 cells. A, flow cytometry demonstrating the marked inhibition of differentiation by antisense-RB, but not by SC-RB. B, quantitation of flow cytometry and cytochemical determination of monocyte-specific esterase (n = 3). *, P < 0.05 relative to the 1,25D3 treatment group and to the 1,25D3 + SC-RB treatment group. C, Western blots showing that antisense RB blocks up-regulation of retinoblastoma protein but does not block up-regulation of CCAAT/enhancer-binding protein ß or vitamin D receptor by 1,25D3. Lane numbers are as defined in B. D, quantitation of three similar experiments. *, P < 0.05 relative to both the 1,25D3 and 1,25D3 + SC-RB treatment groups.

 
C/EBPß Transcription Factor Positively Regulates 1,25D3-Induced Differentiation of HL60 Cells.
C/EBP transcription factors control gene expression by binding to the cognate C/EBP DNA sequence in the promoter regions of the genes that they regulate (43 , 44) . We therefore initiated the study of the role of the C/EBP family of proteins in 1,25D3-induced differentiation by incubating the cells with a great excess of oligonucleotides containing either this DNA element (decoy C/EBP) or its SC sequence version (SC-decoy C/EBP). Although the SC decoy C/EBP had a modest negative effect on 1,25D3-induced differentiation, the effect of decoy C/EBP was significantly (P < 0.05) greater (Fig. 4, A and B)Citation . Western blot analysis of the effects of the decoy on the levels of pRb and VDR showed that neither of these proteins was affected (Fig. 4, C and D)Citation . Thus, inhibition of binding of C/EBPß to its cognate DNA site does not affect the expression of pRb or VDR.



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  		Fig. 4. A, decoy CCAAT/enhancer-binding protein (C/EBP) binding sequence oligonucleotide blocks 1,25-dihydroxyvitamin D3 (1,25D3)-induced differentiation in HL60 cells as shown by flow cytometry. B, quantitation of data shown in A and of cytochemical determination of monocyte-specific esterase activity (n = 3). *, P < 0.05 relative to both the 1,25D3 treatment group and the 1,25D3 + SC decoy C/EBP treatment group. Lane numbers are as defined in B. C, Western blots showing that decoy C/EBP oligonucleotide does not block the up-regulation of retinoblastoma protein or vitamin D receptor induced by 1,25D3. D, quantitation of three blots similar to those shown in C. No significant changes were detected.

 
Previous reports have indicated that hematopoietic cells express several members of the C/EBP family, including C/EBP{alpha} and C/EBPß (21 , 22) . C/EBP{alpha} is implicated in granulopoiesis (45) , whereas C/EBPß is implicated in macrophage function (46 , 47) . Experiments in which AS-C/EBP{alpha} and AS-C/EBPß were used showed that only AS-C/EBPß reduced 1,25D3-induced differentiation, although not to the extent that resulted from the exposure to the decoy C/EBP (Fig. 5)Citation . This difference may be due a greater efficiency of the decoy as compared with the AS strategy, but because we did not test all members of the large C/EBP family (29) , it is possible that another C/EBP protein contributes to monocytic differentiation. However, our findings show that C/EBPß, not C/EBP{alpha}, is the principal member of the C/EBP transcription factor family that contributes to the 1,25D3-induced differentiation of HL60 cells.



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  		Fig. 5. Antisense CCAAT/enhancer-binding protein (C/EBP) ß oligonucleotide, but not antisense C/EBP{alpha} oligonucleotide, blocks 1,25-dihydroxyvitamin D3 (1,25D3)-induced differentiation of HL60 cells (n = 3). *, P < 0.05 relative to the 1,25D3 treatment group and the 1,25D3 + SC-C/EBPß treatment group.

 
Expression of pRb and C/EBPß Is Regulated by VDR.
Having established that the expression of VDR is not under the control of RB or C/EBPß genes, we enquired whether the reverse is true. For this purpose, we used a carboxylic ester antagonist of 1,25D3 (ZK159222) with a minimal agonist activity of 1,25D3 but the ability to block VDR-transmitted signals (48) . As shown in Fig. 6ACitation , ZK159222 had no significant (P < 0.05) differentiation-inducing activity of its own, but when combined with 1,25D3, it markedly inhibited differentiation. Western analysis showed that the expression of both pRb and C/EBPß was markedly decreased by the VDR antagonist (Fig. 6B)Citation . Thus, VDR action is required for the 1,25D3-induced, differentiation-associated, up-regulation of pRb and C/EBPß.



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  		Fig. 6. The 1,25-dihydroxyvitamin D3 (1,25D3) antagonist ZK159222 inhibits 1,25D3-induced differentiation as well as expression of retinoblastoma protein and CCAAT/enhancer-binding protein ß, but not CCAAT/enhancer-binding protein {alpha}. HL60 cells were treated for 24 h with 1,25D3 (10 nM), alone or in combination with ZK159222 (100 nM). *, P < 0.05 compared with treatment with 1,25D3.

 
Three MAPK Pathways Are Upstream of the Expression of pRb and C/EBPß.
Monocytic differentiation of HL60 cells is also influenced by the activity of at least three MAPK pathways (30 , 31 , 33 , 49 , 50) . We therefore determined the effect of inhibition of each of these pathways on the expression of the two proteins that are the focus of these studies, pRb and C/EBPß. The results showed that the inhibition of the extracellular signal-regulated kinase/MAPK and c-Jun-NH2-terminal kinase/MAPK pathways by specific pharmacological inhibitors (51 , 52) , PD98059 and SP600125, respectively, also reduced the extent of differentiation and the expression of pRb and C/EBPß (Fig. 7)Citation . In contrast, the inhibition of the p38/MAPK pathway by its specific inhibitor, SB202190 (53) , increased both the 1,25D3-induced differentiation and the expression of pRb and C/EBPß (Fig. 7)Citation . These data demonstrate that the expression of pRb and C/EBPß is under transcriptional, although probably indirect, control by MAPK pathways, raising the possibility that VDR transmits the signal from MAPK pathways to the pRb·C/EBPß complex, as schematized in Fig. 8Citation .



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  		Fig. 7. The mitogen-activated protein/ERK kinase 1/2 inhibitor PD98059 and the c-Jun-NH2-terminal kinase inhibitor SP600125 reduce 1,25-dihydroxyvitamin D3 (1,25D3)-induced differentiation (A), as well as retinoblastoma protein and CCAAT/enhancer-binding protein ß and mRNA expression (B and D), whereas the p38MAP inhibitor SB202190 has an opposite effect. C and E show quantitation of these experiments. HL60 cells were treated for 48 h with 5 nM 1,25D3, with or without the inhibitors, all at 10 µM concentration. n = 3; *, P < 0.05 compared with treatment with the same concentration of 1,25D3.

 


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  		Fig. 8. A partly hypothetical schematic of the pathways suggested to induce differentiation of HL60 cells by deltanoids. The initial signal provided by 1,25-dihydroxyvitamin D3 is represented as activation (shown by the asterisk) and stabilization of the vitamin D receptor protein, which then heterodimerizes with an isoform of retinoid X receptor and transactivates genes containing its cognate DNA response element, e.g., p21Cip1. One result is the sensitization of the mitogen-activated protein kinase pathways to the ambient stimuli, which include growth- and stress-inducing factors (66) . Subsequently, activation of the activator protein-1 transcription factor may increase transcription of vitamin D receptor (67) and aid the transcription of retinoblastoma and CCAAT/enhancer-binding protein ß genes. This multistep process results in an up-regulation of genes responsible for the monocytic phenotype.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The importance of pRb as a regulator of cell cycle progression achieved, at least in part, by its binding to E2F transcription factors is so well appreciated that it tends to overshadow the fact that pRb has many other potential protein partners (18 , 22) . These proteins include members of the C/EBP transcription factor family, and in this study we focused on the participation the pRb·C/EBPß complex in 1,25D3- induced monocytic differentiation of human leukemia HL60 cells. It is evident that in this system the up-regulation of pRb and C/EBPß expression occurs early in the differentiation process and is required for differentiation and that, although independent of each other, pRb and C/EBPß are both under the control of VDR and MAPK pathways. These findings allow the formulation of a hypothetical "road map" that can explain, in part, the routing of signals through which 1,25D3 and other deltanoids induce monocytic differentiation.

Phenotypic differentiation of deltanoid-induced HL60 cells is known to precede alterations in the cell cycle traverse (33 , 54, 55, 56, 57) . In these cells an increase in early markers of the changing phenotype, such as CD14, the lipopolysaccharide surface receptor characteristic of the macrophage (58 , 59) , or MSE, a cytoplasmic hydrolytic enzyme present in phagocytic cells (58 , 60 , 61) , can be detected in a proportion of cells as early as 8 h after the addition of a deltanoid (Fig. 1)Citation . In contrast, changes in the cell cycle traverse are detectable approximately 30–40 h later (62) . Because the up-regulation of pRb closely approximates the appearance of the early markers of differentiation, this suggests a role in the differentiation process, independent of its cell cycle controls, and this was confirmed by the inhibition of differentiation when pRb expression is down-regulated after the addition of an AS oligonucleotide to pRb (Fig. 3)Citation .

In a previous study we showed that the ratio of highly phosphorylated to hypophosphorylated pRb varied with the strength of the differentiation signal: pRb was highly phosphorylated when the differentiation signal was weak, and no cell cycle effects could be noted (30) . Nonetheless, the weak signal elicited an increase in total pRb and expression of early differentiation markers, suggesting that the phosphorylation status of pRb is more critical for cell cycle control than for differentiation. Whether phosphorylation-induced changes in pRb structure affect its ability to bind C/EBPß to any extent in this system remains to be investigated.

C/EBPß is one of the known partners of pRb in U937 hematopoietic cells (22) . We detected this complex in differentiating HL60 cells (Fig. 2)Citation and observed changes in the levels of C/EBPß and pRb mRNA and proteins that parallel differentiation (Fig. 7)Citation . The approximate equivalence of the magnitude of these changes during differentiation is intriguing. It is consistent with the possibility that the complex has a functional significance, such as transactivation by binding to C/EBPß-dependent genes whose products participate, directly or indirectly, in the process of differentiation of HL60 cells. This possibility is made more likely by the marked inhibition of differentiation when the cells were exposed to a "decoy" C/EBP element, which can sequester the C/EBP element-binding proteins (Fig. 4)Citation .

It is important to realize that hematopoietic cells in general, but especially HL60 cells, have a very low efficiency of transfection. This makes them unsuitable for introduction of plasmids that can alter cellular activities (63, 64, 65) . However, they take up DNA oligonucleotides without treatment such as lipofection or electroporation that can destroy cellular membranes. Because prolonged exposure to the oligonucleotides can also be cytotoxic, the experiments were limited to 24 h of exposure to the oligonucleotide, at which time cell viability was not impaired (data not shown). Consequently, the effects of the oligonucleotides on the cell cycle traverse could not be studied in the HL60 cell-deltanoid system in this short time frame.

VDR has a pivotal role in deltanoid-induced differentiation because cells that do not express VDR are unresponsive to these compounds. We demonstrate here that a carboxylic ester derivative of 1,25D3 with antagonist action on VDR markedly inhibits not only differentiation but also expression of pRb and C/EBPß (Fig. 6)Citation . Furthermore, pRb and C/EBPß are also under the control (although probably indirect) of at least three MAPK pathways, which are normally activated by growth factors, cytokines, and stress (66) and appear to be further sensitized by the presence of deltanoids (30, 31, 32, 33) . The effects (up- or down-regulation) of these pathways on monocytic differentiation are paralleled by changes in the expression of pRb and C/EBPß (Fig. 7)Citation . Interestingly, whereas the extracellular signal-regulated kinase and c-Jun-NH2-terminal kinase MAPK pathways enhance 1,25D3-induced differentiation, the p38MAPK pathway has an opposite effect (Fig. 7)Citation , suggesting that it may dampen and thus control the differentiation processes by negative feedback. The nature of this putative feedback requires further study.

Although important gaps in knowledge still exist, a provisional schematic can now be constructed in which the pRb·C/EBPß complex has an important and perhaps central role in deltanoid-induced differentiation, as shown in Fig. 8Citation . This role may be not unlike the functions of the pRb·E2F complexes in the control of cell cycle traverse, so critical to the proliferation of normal and neoplastic cells.


   ACKNOWLEDGMENTS
 
We thank Dr. A. Steinmeyer (Heinrich-Heine-Universitat, Dusseldorf, Germany) for the gift of ZK159222 and Dr. Milan Uskokvic (BioXell, Inc., Nutley, NJ) for a generous supply of 1,25D3. We are also grateful to Dr. Robert Murray (University of Toronto, Toronto, Canada) for comments on the manuscript and Dr. Xuening Wang (NJMS) for help in its preparation.


   FOOTNOTES
 
Grant support: New Jersey Commission for Cancer Research and Grant RO1-CA44722 (to G. P. Studzinski) from the National Cancer Institute, NIH.

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.

Requests for reprints: George P. Studzinski, Department of Pathology and Laboratory Medicine, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, New Jersey 07013. Phone: (973) 972-5869, Fax: (973) 972-7293; E-Mail: studzins{at}umdnj.edu

Received 9/25/03. Revised 10/24/03. Accepted 10/31/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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