Antisense Oligonucleotides Targeted against Protein Kinase Cbeta and Cbeta II Block 1,25-(OH)2D3-induced Differentiation*

Robert U. SimpsonDagger §, Timothy D. O'ConnellDagger , Quintin PanDagger parallel , Judy NewhouseDagger , and Martha J. SomermanDagger **

From the Dagger  Department of Pharmacology and ** Department of Periodontics/Prevention/Geriatrics, the University of Michigan, Ann Arbor, Michigan 48109-0632

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

It is now recognized that protein kinase C (PKC) plays a critical role in 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) promotion of HL-60 cell differentiation. In this study, the effects of phosphorothioate antisense oligonucleotides directed against PKCalpha , PKCbeta , PKCbeta I, and PKCbeta II on HL-60 promyelocyte cell differentiation and proliferation were examined. Cellular differentiation was determined by nonspecific esterase activity, nitro blue tetrazolium reduction, and CD14 surface antigen expression. Differentiation promoted by 1,25-(OH)2D3 (20 nM for 48 h) was inhibited similarly in cells treated with PKCbeta antisense (30 µM) 24 h prior to or at the same time as hormone treatment (86 ± 9% inhibition; n = 4 versus 82 ± 8% inhibition; n = 4 (mean ± S.E.), respectively). In contrast, cells treated with PKCbeta antisense 24 h after 1,25-(OH)2D3 were unaffected and fully differentiated. PKCalpha antisense did not block 1,25-(OH)2D3 promotion of HL-60 cell differentiation. Next, the ability of PKCbeta I- and PKCbeta II-specific antisense oligonucleotides to block 1,25-(OH)2D3 promotion of cell differentiation was examined. PKCbeta II antisense (30 µM) completely blocked CD14 expression induced by 1,25-(OH)2D3, whereas PKCbeta I antisense had little effect. Interestingly, PKCbeta II antisense blocked differentiation by 87 ± 7% (n = 2, mean ± S.D.) but had no effect on 1,25-(OH)2D3 inhibition of cellular proliferation. These results indicate that the effects of 1,25-(OH)2D3 on HL-60 cell differentiation and proliferation can be dissociated by blocking PKCbeta II expression.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The hormone 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)1 regulates the growth and maturation of numerous organs and cell types. 1,25-(OH)2D3 is involved in the control of calcium and phosphorus homeostasis, muscle function, immunity, endocrine secretions, and neurotransmission (1). It is accepted that, in part, this hormone alters cell function by enhancing or repressing expression of specific genes (2, 3). Other studies have revealed that 1,25-(OH)2D3 regulates cellular processes without altering gene expression (4). These observations suggest that nongenomic effects of the hormone occur and result in the rapid alteration of cell membrane phospholipid metabolism and intracellular calcium concentrations (5, 6). Although the exact mechanism by which 1,25-(OH)2D3 promotes HL-60 cell differentiation is not fully understood, a number of studies from our laboratory and others have implicated protein kinase C (PKC) as a critical component of this process (7-9).

PKC is a family of serine-threonine protein kinases, which play major roles in regulation of many cellular processes. To date, 11 PKC isoenzymes have been characterized and classified into three groups based on their structure and activation requirements (10, 11). The classical PKCs, PKCalpha , PKCbeta I, PKCbeta II, and PKCgamma , require calcium for activation. A second class of PKCs has been termed the novel PKCs and consist of PKCdelta , PKCepsilon , PKCeta , and PKCtheta (11). These novel PKCs do not have a calcium binding motif, and therefore calcium is not required for activation. The third class of PKCs are called the atypical PKCs and include PKClambda , PKCµ, and PKCzeta . These PKCs differ significantly in structure to the other PKCs. Furthermore, atypical PKCs do not respond to phorbol ester activation.

The importance of PKC in 1,25-(OH)2D3 promotion of HL-60 cells along the monocyte/macrophage pathway is now appreciated. Our laboratory reported that 1,25-(OH)2D3 increases PKC levels in HL-60 cells (7). Additionally, we found that classical inhibitors of PKC, H-7 and staturosporine, block the ability of 1,25-(OH)2D3 to promote HL-60 cell differentiation (12, 13). Using similar PKC inhibitors, PKC activation by 1,25-(OH)2D3 has been shown to be involved in skin, heart, skeletal muscle, and renal cell gene expression and function (14-17). Unfortunately, such chemical inhibitors are of little use in determining isoenzyme specificity for a cellular transduction mechanism. Recent studies have used overexpression and antisense techniques to provide evidence that PKCbeta is, to some extent, the isoenzyme involved in 1,25-(OH)2D3 promotion of HL-60 cell differentiation (18, 19). In this study, we showed that increased PKCbeta II levels by 1,25-(OH)2D3 is required to promote HL-60 cell differentiation. Interestingly, PKCbeta II antisense had no effect on 1,25-(OH)2D3 inhibition of HL-60 cell proliferation. Our report shows that increases in PKCbeta II levels and activation are important events in 1,25-(OH)2D3 promotion of cell differentiation. Moreover, we suggest that the events leading to cellular differentiation most likely require protein phosphorylation.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Chemicals-- 1,25-(OH)2D3 was purchased from Tetrionics Inc. (Madison, WI). Vitamin D3 metabolite purity and structural integrity were confirmed by high performance liquid chromatography and UV spectroscopy. All other reagents were reagent grade or better.

Cell Culture-- HL-60 promyelocytic leukemia cells were obtained from American Type Culture Collection (Rockville, MD) and cultured in RPMI 1640 medium supplemented with 10% horse serum, 1000 units/ml penicillin G, and 0.5 mg/ml streptomycin. Cells were incubated at 37 °C in a humidified atmosphere with 5% CO2. Cells used in this study were from passages 21-45. All experiments were initiated with cells in log phase growth at 2 × 105 cells/ml and then allowed to equilibrate in growth medium for 24 h prior to any treatment. Cellular differentiation was assessed by nonspecific esterase activity, nitro blue tetrazolium dye reduction, and CD14 surface antigen expression (7, 12, 36).

Oligonucleotides-- Phosphorothioate oligonucleotides were synthesized by the DNA Synthesis Center at the University of Michigan. PKCbeta antisense was designed to interact with bases +4 to +18 of the PKCbeta mRNA. PKCbeta sense had the complementary sequence of the same region. PKCbeta oligonucleotides used were: PKCbeta sense, 5'-GCT GAC CCG GCT GCG-3'; PKCbeta antisense, 5'-CGC AGC CGG GTC AGC-3'. PKCalpha antisense was designed to interact with bases +6 to +20 of the PKCalpha mRNA. PKCalpha sense had the complementary sequence over the same region. PKCalpha oligonucleotides used were: PKCalpha sense, 5'-TCG GGG GGG ACC ATG-3'; PKCalpha antisense, 5'-CAT GGT CCC CCC CGA-3'. PKCbeta I and PKCbeta II specific antisenses were designed to interact with bases +1942 to +1956 that exist 3' from the splice site. PKCbeta I sense and PKCbeta II sense had the complementary sequences to its respectful antisense pair. PKCbeta I antisense was: 5'-GTT TTA AGC ATT TCG-3'; PKCbeta II antisense was: 5'-GTT GGA GGT GTC TCT-3'.

Western Blot Analysis of PKCbeta and PKCalpha Protein Levels-- Cells were washed two times with phosphate-buffered saline then resuspended in lysis buffer (0.2 M Tris, 0.5 mM EGTA, 0.5 mM EDTA, 0.5% Triton X-100, 100 mM leupeptin, 0.4 mM phenylmethylsulfonyl fluoride, pH 7.5) and homogenized using a Dounce homogenizer. Protein content of total cell homogenates was determined by the Bradford protein assay (37). Equal amounts of protein from each condition were run on a 10% polyacrylamide gel, and proteins were subsequently transferred to Immobilon paper (Millipore, Bedford, MA). The blot was blocked with buffer containing 1% bovine serum albumin (10 mM Tris, 0.1% Tween 20, and 1% bovine serum albumin, pH 7.4). It was then probed for 2 h with primary antibodies (PKCbeta , PKCbeta I, PKCbeta II, and PKCalpha antibodies; Life Technologies Inc.), then washed three times with blocking buffer and incubated for 1.5 h with a secondary antibody conjugated with horseradish peroxidase (Sigma). The blot was then washed five times with Tween-TBS (10 mM Tris and 0.2% Tween 20, pH 7.4). Finally, it was developed using enhanced chemiluminescence (Amersham Pharmacia Biotech) and exposed to x-ray film.

Cell Proliferation Assay-- Cell number was determined using a Coulter (Coulter Electronics, Hialeah, FL) model Zf cell counter. Cell viability was determined using trypan blue dye exclusion.

Statistical Analysis of Data-- Differences between 1,25-(OH)2D3 treated cells and untreated cells for all assays were evaluated by unpaired Student's t test.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Effect of PKCbeta Antisense on 1,25-(OH)2D3 Induction of PKCbeta Levels-- HL-60 cells were treated with control (0.1% ethanol) or 20 nM 1,25-(OH)2D3 and PKCbeta sense or antisense for 48 h. PKCbeta protein levels were determined by Western blot analysis (Fig. 1). Cells treated with 1,25-(OH)2D3 in the presence of PKCbeta sense oligonucleotide showed similar increases in PKCbeta levels as compared with cells exposed to 1,25-(OH)2D3 alone. Importantly, cells treated with PKCbeta antisense (lane 4) exhibited a mark inhibition in 1,25-(OH)2D3 induction of PKCbeta levels. PKCbeta levels were decreased by 81 ± 9% (mean ± S.E.) relative to sense or oligonucleotide free cultures. The antibody used to detect PKCbeta in this experiment was not specific for the splice isoenzymes beta I or beta II. As seen in Fig. 1, untreated (control) cells routinely exhibited minimal levels of PKCbeta . Furthermore, PKCbeta levels remain unchanged in uninduced (control) cells even after 48 h of PKCbeta antisense treatment (n = 13, lanes 1 and 2, Fig. 1). This observation is expected, because PKCbeta has a half-life of greater than 70 h. Therefore, blocking translation with PKCbeta antisense would not greatly influence existing levels of PKCbeta . As shown in Fig. 2, PKCbeta levels were increased within 24 h of 1,25-(OH)2D3 treatment. Furthermore, PKCbeta antisense significantly blocked 1,25-(OH)2D3 induction of PKCbeta levels at 24 and 48 h of hormone treatment. Therefore, these results demonstrate that the PKCbeta antisense oligonucleotide is able to block the induction of PKCbeta levels by 1,25-(OH)2D3.


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Fig. 1.   Effects of PKCbeta antisense on 1,25-(OH)2D3-induced increases of PKCbeta protein levels. HL-60 cells were treated with control or 20 nM 1,25-(OH)2D3 in the presence of 30 µM PKCbeta sense or PKCbeta antisense for 48 h. PKCbeta levels were visualized by Western blot analysis and quantified by densitometry. In seven experiments, PKCbeta antisense significantly (p < 0.01) blocked 1,25-(OH)2D3 induction of PKCbeta levels.


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Fig. 2.   Time course of PKCbeta antisense treatment on 1,25-(OH)2D3-induced increases of PKCbeta protein levels. HL-60 cells were treated with 20 nM 1,25-(OH)2D3 for 1, 6, 24, or 48 h in the presence of 30 µM PKCbeta sense or PKCbeta antisense. In lane 1, cells were treated with vehicle (Control) for 48 h. Cells were harvested at each time point, and PKCbeta levels were visualized by Western blot analysis.

Specificity of PKCbeta Antisense to Inhibit 1,25-(OH)2D3 Induction of PKCbeta Protein Levels-- HL-60 cells were treated with 20 nM 1,25-(OH)2D3 alone (C) or with PKCalpha sense, PKCalpha antisense, PKCbeta sense, or PKCbeta antisense (30 µM amount of either oligonucleotide) for 48 h. PKCbeta levels were determined by Western blot analysis (Fig. 3A). PKCbeta levels in cells treated with 1,25-(OH)2D3 and PKCalpha sense, PKCalpha antisense, or PKCbeta sense were not significantly different from cells treated with 1,25-(OH)2D3 alone. As expected, PKCbeta antisense was able to inhibit the induction of PKCbeta by 1,25-(OH)2D3 (lane 5). Moreover, as shown in Fig. 3B, PKCalpha antisense was able to specifically block 1,25-(OH)2D3 enhancement of PKCalpha levels. Importantly, PKCbeta antisense had no effect on 1,25-(OH)2D3 induction of PKCalpha levels. These results demonstrate that PKCbeta antisense has specificity in blocking 1,25-(OH)2D3-induced increases of PKCbeta . Western blot analysis of PKCbeta I and PKCbeta II splice isoenzymes was performed using specific antibodies. PKCbeta II protein levels were detectable using these specific antibodies, whereas PKCbeta I levels were not detectible. This observation in HL-60 cells is similar to ones reported previously (20, 21). Also, the PKCalpha antisense oligonucleotide at concentrations up to 60 µM had no effect on 1,25-(OH)2D3 promotion of cell differentiation (data not shown).


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Fig. 3.   Specificity of PKCalpha and PKCbeta antisense. A, PKCbeta protein levels. HL-60 cells were treated with 20 nM 1,25-(OH)2D3 alone (C) or 20 nM 1,25-(OH)2D3 with 30 µM PKCalpha sense, PKCalpha antisense, PKCbeta sense, or PKCbeta antisense for 48 h. B, PKCalpha protein levels. HL-60 cells were treated with 20 nM 1,25-(OH)2D3 alone (C) or 20 nM 1,25-(OH)2D3 with 30 µM PKCalpha sense, PKCalpha antisense, or PKCbeta antisense for 48 h. PKCalpha and PKCbeta levels were visualized by Western blot analysis. This figure is representative of three independent experiments.

Dose Response of PKCbeta Antisense to Inhibit 1,25-(OH)2D3 Induction of PKCbeta Levels and Cell Differentiation-- HL-60 cells were treated with 20 nM 1,25-(OH)2D3 and either 30 µM PKCbeta sense or 1, 10, or 30 µM PKCbeta antisense for 48 h. PKCbeta levels were determined by Western blot analysis (Fig. 4A). As shown in Fig. 4A, a dose-dependent decrease in PKCbeta levels was observed with increasing concentrations of PKCbeta antisense. In this experiment an 85% decrease, as determined by scanning densitometry, in PKCbeta levels was observed with 30 µM PKCbeta antisense. Next, the effects of antisense constructs on HL-60 cell differentiation and the importance of the time of PKCbeta antisense addition, relative to 1,25-(OH)2D3 treatment, were also examined. HL-60 cells were treated with PKCbeta sense or PKCbeta antisense 24 h prior to (open symbols) or at the same time (closed symbols) as 20 nM 1,25-(OH)2D3. Cell differentiation was determined by nitro blue tetrazolium dye reduction (circles) and nonspecific esterase activity (squares) (Fig. 4B). HL-60 cells treated with 1,25-(OH)2D3 in the absence of oligonucleotide treatment were induced to differentiate to the same extent as cells pretreated or co-treated with PKCbeta sense (data not shown). Differentiation promoted by 1,25-(OH)2D3 was inhibited by 86 ± 9% in cells pretreated with PKCbeta antisense (30 µM) and 82 ± 8% in cells co-treated with PKCbeta antisense (Fig. 4B). Therefore, it is likely that the action of the antisense construct is not to lower existing PKCbeta levels but to block 1,25-(OH)2D3-induced increases in PKCbeta synthesis. However, if cells were first treated with 1,25-(OH)2D3 for 24 h prior to PKCbeta antisense, antisense treatment was ineffective in blocking 1,25-(OH)2D3 promotion of cell differentiation (hatched circles and squares; Fig. 4B). This observation suggests that 1,25-(OH)2D3 has induced sufficient de novo synthesis of PKCbeta within 24 h to render the antisense PKCbeta construct impotent. Thus, these experiments reveal that a relevant and required action of 1,25-(OH)2D3 in promoting HL-60 cell differentiation is to up-regulate PKCbeta levels by increasing the synthesis of the enzyme.


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Fig. 4.   Dose response of PKCbeta antisense. A, 1,25-(OH)2D3-induced increases of PKCbeta protein levels. HL-60 cells were treated with 20 nM 1,25-(OH)2D3 and 30 µM PKCbeta sense or 1, 10, or 30 µM PKCbeta antisense for 48 h. PKCbeta levels were determined by Western blot analysis. B, 1,25-(OH)2D3 induction of cell differentiation. HL-60 cells were treated with 30 µM PKCbeta sense or 1, 10, or 30 µM PKCbeta antisense 24 h prior to (open symbols) at the same time as (closed symbols) or 24 h after (hatched symbols) the addition of 20 nM 1,25-(OH)2D3. Cell differentiation was determined by nitro blue tetrazolium dye reduction (circles) and nonspecific esterase activity (squares). Data are presented as percent of differentiated cells with the control value subtracted and represents the mean ± S.E. of three independent determinations.

Effects of PKCbeta I and PKCbeta II Antisense on HL-60 Cell Differentiation-- HL-60 cells were treated with vehicle, 20 nM 1,25-(OH)2D3, or 20 nM 1,25-(OH)2D3 and PKCbeta sense, PKCbeta antisense, PKCbeta I sense, PKCbeta I antisense, PKCbeta II sense, or PKCbeta II antisense (30 µM) for 72 h, and cell differentiation was determined by CD14 surface antigen expression using flow cytometry (Fig. 5). CD14 is a cell surface marker of mature monocytes/macrophages. Treatment with 1,25-(OH)2D3 significantly increased cell differentiation as shown by the substantial increase in CD14 expression (Fig. 5A). PKCbeta sense did not affect 1,25-(OH)2D3-induced expression of CD14, whereas PKCbeta antisense completely blocked the ability of 1,25-(OH)2D3 to increase CD14 expression. This is consistent with all previous observations and again demonstrates that PKCbeta is essential for 1,25-(OH)2D3-induced differentiation of HL-60 cells. In Fig. 5, C and D, the potency of antisense constructs designed to hybridize specifically with PKCbeta I and PKCbeta II was examined. A complete block of 1,25-(OH)2D3-induced CD14 expression was observed with the PKCbeta II-specific antisense oligonucleotide (Fig. 5C). In contrast, PKCbeta I-specific antisense failed to reverse the enhanced expression of CD14 by 1,25-(OH)2D3 (Fig. 5D).


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Fig. 5.   Immunoflow cytometry analysis of CD14 surface antigen expression. A, control versus 20 nM 1,25-(OH)2D3-treated cells. B, 20 nM 1,25-(OH)2D3 and 30 µM PKCbeta sense versus 20 nM 1,25-(OH)2D3 and 30 µM PKCbeta antisense. C, 20 nM 1,25-(OH)2D3 and 30 µM PKCbeta II sense versus 20 nM 1,25-(OH)2D3 and 30 µM PKCbeta II antisense. D, 20 nM 1,25-(OH)2D3 and 30 µM PKCbeta I sense versus 20 nM 1,25-(OH)2D3 and 30 µM PKCbeta I antisense. Cells were harvested after 48 h of treatment and incubated with fluorescein-labeled anti-CD14 antibody. The intensity of CD14 expression by individual cells are presented as histograms. This figure is representative of three independent experiments.

Effects of PKCbeta II Antisense on Cell Proliferation-- Interestingly, PKCbeta II antisense did not block 1,25-(OH)2D3 inhibition of HL-60 cell proliferation (Fig. 6). Thus, these data show that blocking 1,25-(OH)2D3-stimulated increase in PKCbeta II decreased the induction of cell differentiation by 80% but had no effect on 1,25-(OH)2D3 inhibition of cell proliferation. Similar results were obtained with the less specific PKCbeta antisense construct (data not shown).


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Fig. 6.   Effects of PKCbeta II antisense on 1,25-(OH)2D3 inhibition of HL-60 cell proliferation. HL-60 cells were treated with control, 20 nM 1,25-(OH)2D3, or 20 nM 1,25-(OH)2D3 in the presence of 30 µM PKCbeta II sense or PKCbeta II antisense. Cell number was determined by Coulter counter on the indicated days. Cell number was significantly (p < 0.05) different in control cells versus 1,25D3 + beta IIs or 1,25D3 + beta IIas at days 4, 5, and 6. No significant (p > 0.05) difference in cell number was observed between 1,25D3 + beta IIs and 1,25D3 + beta IIas over the entire time course.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

1,25-(OH)2D3 affects the growth and differentiation of numerous cell types (22-26). Relevant to this report HL-60 cells have been shown to differentiate into monocytes-macrophages (24) and osteoclast-like cells (23) upon exposure to 1,25-(OH)2D3. Expression of several genes including c-myc, c-fos, and PKCalpha , PKCbeta , and PKCgamma are regulated prior to the appearance of the mature monocytic-macrophage phenotype (7-9, 28). c-myc gene expression is decreased, c-fos gene expression is transiently increased, and PKC levels are increased in HL-60 cells during the process of cellular differentiation (25, 29, 30). Considering the nature of these early events and the accepted importance of these gene products in cell signaling and growth, it is likely that regulation of these genes is critical for induced HL-60 cell differentiation.

Recent reports revealed that the 1,25-(OH)2D3 receptor (vitamin D receptor) is a substrate for PKCbeta and that phosphorylation of vitamin D receptor is important for controlling osteocalcin expression (3, 27). Such studies support and extend the possible roles PKCs have in modulating 1,25-(OH)2D3's actions. Transcriptional response elements for 1,25-(OH)2D3 have also been identified. Interestingly, the response element for 1,25-(OH)2D3 in the osteocalcin gene contains a phorbol ester response element (31, 32). One factor that interacts with this AP-1 sequence is a heterodimer made up of c-fos and c-jun. PKC-directed phosphorylation of c-fos and c-jun regulates their AP-1 binding activity (33). However, the precise molecular nature of the interaction between the 1,25-(OH)2D3 signal transduction pathway and PKC for regulation of gene expression is still not clear. Several nuclear proteins have been shown to be phosphorylated by PKC during the course of myeloid cell differentiation (34). Our laboratory reported that 10 nuclear proteins undergo phosphorylation state changes within 6-40 h of 1,25-(OH)2D3 treatment (34). We identified several of these proteins as nuclear matrix or DNA packaging proteins, including several histones and lamin B. Therefore, PKCs act as regulators of nuclear events and may be intimately involved in the transduction of the 1,25-(OH)2D3 signal ultimately regulating gene expression and HL-60 cell differentiation.

Increasing evidence exists to indicate that PKCbeta plays an important role in 1,25-(OH)2D3 promotion of HL-60 cell differentiation. A variant HL-60 cell line (HL-525) lacking basal levels of PKCbeta is resistant to phorbol ester-induced differentiation (18). However, susceptibility to phorbol ester differentiation was restored if HL-525 cells were transfected to overexpress PKCbeta . Additionally, phorbol 12-myristate 13-acetate resistance of HL-525 cells was reversed by pretreating with 1,25-(OH)2D3, which increased PKCbeta levels (18). Also, it was shown that a 25-mer PKCbeta antisense construct different from the one used here was capable of partially blocking (averaging approx 30%) 1,25-(OH)2D3's induction of cell differentiation (19). Although a partial inhibition of 1,25-(OH)2D3-promoted cell differentiation was observed using their antisense construct, it had little effect on 1,25-(OH)2D3 inhibition of cell proliferation. In our study, novel 15-mer PKCbeta and PKCbeta II antisense constructs were found to inhibit 1,25-(OH)2D3 promotion of cell differentiation by 80-90%. However, these antisense oligonucleotides had no effect on 1,25-(OH)2D3's ability to inhibit cell proliferation. Moreover, reduction of basal levels of PKCbeta was not required for PKCbeta antisense to inhibit 1,25-(OH)2D3 promotion of cell differentiation. This result suggests that blocking de novo synthesis of PKCbeta is the mechanism of action for the antisense construct. We demonstrated that PKCbeta II is uniquely responsible for 1,25-(OH)2D3 promotion of cell differentiation. There is controversy as to whether PKCbeta I is expressed in HL-60 cells. In all reports, PKCbeta I levels in unstimulated HL-60 cells is significantly lower than PKCbeta II levels (19-21, 35). In this study, we failed to detect measurable levels of PKCbeta I. This finding is in agreement with several reports (20, 21). However, others have shown, using different antibodies or Northern blot analysis, that 1,25-(OH)2D3 increased PKCbeta I protein levels or mRNA levels (19, 35).

The findings reported here indicate that PKCbeta II specifically participates in the signal transduction mechanisms employed by 1,25-(OH)2D3 to promote HL-60 cell differentiation. Interestingly, we found a direct correlation between the quantitative lowering of PKCbeta II protein levels and the degree of induced differentiation. The correlation between the increased levels of PKCbeta induced by 1,25-(OH)2D3 and the extent of cellular differentiation (7-9) suggest that there are not spare PKCbeta s in these cells. Thus, we suggest that the levels of PKCbeta are stoichometrically related to promotion of differentiation. This study provides clear and convincing evidence that promotion of cell differentiation and inhibition of cell proliferation are two distinct processes by 1,25-(OH)2D3 that can be disassociated by blocking the expression of a single gene, PKCbeta II. To date, several analogs of 1,25-(OH)2D3 have been developed that are selective at affecting calcium mobilization and promoting terminal cellular differentiation. Our study suggests that it may be possible to further separate the actions of 1,25-(OH)2D3 into its capacity to promote cellular differentiation versus its capacity to inhibit cell proliferation.

    ACKNOWLEDGEMENT

We thank Li Huang for her technical efforts and expertise.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant DE 10337 (to R. U. S. and M. J. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. of Pharmacology, 2301D MSRB III, University of Michigan, Ann Arbor, MI 48109. Tel.: 734-763-3255; Fax: 734-763-4450; E-mail: robsim{at}umich.edu.

Current address: Cardiovascular Institute and Division of Cardiology, San Francisco Veterans Hospital, San Francisco, CA 94105.

parallel Newton-Loeb Fund/University of Michigan Cancer Center Predoctoral Fellow.

1 The abbreviations used are: 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; PKC, protein kinase C.

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Abstract
Introduction
Procedures
Results
Discussion
References

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