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

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 PKCα, PKCβ, PKCβI, and PKCβII 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 PKCβ antisense (30 μm) 24 h prior to or at the same time as hormone treatment (86 ± 9% inhibition; n = 4versus 82 ± 8% inhibition; n = 4 (mean ± S.E.), respectively). In contrast, cells treated with PKCβ antisense 24 h after 1,25-(OH)2D3were unaffected and fully differentiated. PKCα antisense did not block 1,25-(OH)2D3 promotion of HL-60 cell differentiation. Next, the ability of PKCβI- and PKCβII-specific antisense oligonucleotides to block 1,25-(OH)2D3 promotion of cell differentiation was examined. PKCβII antisense (30 μm) completely blocked CD14 expression induced by 1,25-(OH)2D3, whereas PKCβI antisense had little effect. Interestingly, PKCβII antisense blocked differentiation by 87 ± 7% (n = 2, mean ± S.D.) but had no effect on 1,25-(OH)2D3inhibition 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 PKCβII expression.

The hormone 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 ) 1 regulates the growth and maturation of numerous organs and cell types. 1,25-(OH) 2 D 3 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) 2 D 3 regulates cellular processes without altering gene expression (4). These observations sug-gest 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) 2 D 3 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)(8)(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, PKC␣, PKC␤I, PKC␤II, and PKC␥, require calcium for activation. A second class of PKCs has been termed the novel PKCs and consist of PKC␦, PKC⑀, PKC, and PKC (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 PKC, PKC, and PKC. 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) 2 D 3 promotion of HL-60 cells along the monocyte/macrophage pathway is now appreciated. Our laboratory reported that 1,25-(OH) 2 D 3 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) 2 D 3 to promote HL-60 cell differentiation (12,13). Using similar PKC inhibitors, PKC activation by 1,25-(OH) 2 D 3 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 PKC␤ is, to some extent, the isoenzyme involved in 1,25-(OH) 2 D 3 promotion of HL-60 cell differentiation (18,19). In this study, we showed that increased PKC␤II levels by 1,25-(OH) 2 D 3 is required to promote HL-60 cell differentiation. Interestingly, PKC␤II antisense had no effect on 1,25-(OH) 2 D 3 inhibition of HL-60 cell proliferation. Our report shows that increases in PKC␤II levels and activation are important events in 1,25-(OH) 2 D 3 promotion of cell differentiation. Moreover, we suggest that the events leading to cellular differentiation most likely require protein phosphorylation.

EXPERIMENTAL PROCEDURES
Chemicals-1,25-(OH) 2 D 3 was purchased from Tetrionics Inc. (Madison, WI). Vitamin D 3 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 * 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. This 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: units/ml penicillin G, and 0.5 mg/ml streptomycin. Cells were incubated at 37°C in a humidified atmosphere with 5% CO 2 . Cells used in this study were from passages 21-45. All experiments were initiated with cells in log phase growth at 2 ϫ 10 5 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. PKC␤ antisense was designed to interact with bases ϩ4 to ϩ18 of the PKC␤ mRNA. PKC␤ sense had the complementary sequence of the same region. PKC␤ oligonucleotides used were: PKC␤ sense, 5Ј-GCT GAC CCG GCT GCG-3Ј; PKC␤ antisense, 5Ј-CGC AGC CGG GTC AGC-3Ј. PKC␣ antisense was designed to interact with bases ϩ6 to ϩ20 of the PKC␣ mRNA. PKC␣ sense had the complementary sequence over the same region. PKC␣ oligonucleotides used were: PKC␣ sense, 5Ј-TCG GGG GGG ACC ATG-3Ј; PKC␣ antisense, 5Ј-CAT GGT CCC CCC CGA-3Ј. PKC␤I and PKC␤II specific antisenses were designed to interact with bases ϩ1942 to ϩ1956 that exist 3Ј from the splice site. PKC␤I sense and PKC␤II sense had the complementary sequences to its respectful antisense pair. PKC␤I antisense was: 5Ј-GTT TTA AGC ATT TCG-3Ј; PKC␤II antisense was: 5Ј-GTT GGA GGT GTC TCT-3Ј.
Western Blot Analysis of PKC␤ and PKC␣ 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 (PKC␤, PKC␤I, PKC␤II, and PKC␣ 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) 2 D 3 treated cells and untreated cells for all assays were evaluated by unpaired Student's t test.

Effect of PKC␤ Antisense on 1,25-(OH) 2 D 3 Induction of PKC␤
Levels-HL-60 cells were treated with control (0.1% ethanol) or 20 nM 1,25-(OH) 2 D 3 and PKC␤ sense or antisense for 48 h. PKC␤ protein levels were determined by Western blot analysis (Fig. 1). Cells treated with 1,25-(OH) 2 D 3 in the presence of PKC␤ sense oligonucleotide showed similar increases in PKC␤ levels as compared with cells exposed to 1,25-(OH) 2 D 3 alone. Importantly, cells treated with PKC␤ antisense (lane 4) exhibited a mark inhibition in 1,25-(OH) 2 D 3 induction of PKC␤ levels. PKC␤ levels were decreased by 81 Ϯ 9% (mean Ϯ S.E.) relative to sense or oligonucleotide free cultures. The antibody used to detect PKC␤ in this experiment was not specific for the splice isoenzymes ␤I or ␤II. As seen in Fig. 1, untreated (control) cells routinely exhibited minimal levels of PKC␤. Furthermore, PKC␤ levels remain unchanged in uninduced (control) cells even after 48 h of PKC␤ antisense treatment (n ϭ 13, lanes 1 and 2, Fig. 1). This observation is expected, because PKC␤ has a half-life of greater than 70 h. Therefore, blocking translation with PKC␤ antisense would not greatly influence existing levels of PKC␤. As shown in Fig. 2, PKC␤ levels were increased within 24 h of 1,25-(OH) 2 D 3 treatment. Furthermore, PKC␤ antisense significantly blocked 1,25-(OH) 2 D 3 induction of PKC␤ levels at 24 and 48 h of hormone treatment. Therefore, these results demonstrate that the PKC␤ antisense oligonucleotide is able to block the induction of PKC␤ levels by 1,25-(OH) 2 D 3 .
Specificity of PKC␤ Antisense to Inhibit 1,25-(OH) 2 D 3 Induction of PKC␤ Protein Levels-HL-60 cells were treated with 20 nM 1,25-(OH) 2 D 3 alone (C) or with PKC␣ sense, PKC␣ antisense, PKC␤ sense, or PKC␤ antisense (30 M amount of either oligonucleotide) for 48 h. PKC␤ levels were determined by Western blot analysis (Fig. 3A). PKC␤ levels in cells treated with 1,25-(OH) 2 D 3 and PKC␣ sense, PKC␣ antisense, or PKC␤ sense were not significantly different from cells treated with 1,25-(OH) 2 D 3 alone. As expected, PKC␤ antisense was able to inhibit the induction of PKC␤ by 1,25-(OH) 2 D 3 (lane 5). Moreover, as shown in Fig. 3B, PKC␣ antisense was able to specifically block 1,25-(OH) 2 D 3 enhancement of PKC␣ levels. Importantly, PKC␤ antisense had no effect on 1,25-(OH) 2 D 3 induction of PKC␣ levels. These results demonstrate that PKC␤ antisense has specificity in blocking 1,25-(OH) 2 D 3 -induced increases of PKC␤. Western blot analysis of PKC␤I and PKC␤II splice isoenzymes was performed using specific antibodies. PKC␤II protein levels were detectable using these specific antibodies, whereas PKC␤I levels were not detectible. This observation in HL-60 cells is similar to ones reported previously (20,21). Also, the PKC␣ antisense oligonucleotide at concentrations up to 60 M had no effect on 1,25-(OH) 2 (Fig. 4A). As shown in Fig. 4A, a dose-dependent decrease in PKC␤ levels was observed with increasing concentrations of PKC␤ antisense. In this experiment an 85% decrease, as determined by scanning densitometry, in PKC␤ levels was observed with 30 M PKC␤ antisense. Next, the effects of antisense constructs on HL-60 cell differentiation and the importance of the time of PKC␤ antisense addition, relative to 1,25-(OH) 2 D 3 treatment, were also examined. HL-60 cells were treated with PKC␤ sense or PKC␤ antisense 24 h prior to (open symbols) or at the same time (closed symbols) as 20 nM 1,25-(OH) 2 D 3 . 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) 2 D 3 in the absence of oligonucleotide treatment were induced to differentiate to the same extent as cells pretreated or co-treated with PKC␤ sense (data not shown). Differentiation promoted by 1,25-(OH) 2 D 3 was inhibited by 86 Ϯ 9% in cells pretreated with PKC␤ antisense (30 M) and 82 Ϯ 8% in cells co-treated with PKC␤ antisense (Fig.  4B). Therefore, it is likely that the action of the antisense construct is not to lower existing PKC␤ levels but to block 1,25-(OH) 2 D 3 -induced increases in PKC␤ synthesis. However, if cells were first treated with 1,25-(OH) 2 D 3 for 24 h prior to PKC␤ antisense, antisense treatment was ineffective in blocking 1,25-(OH) 2 D 3 promotion of cell differentiation (hatched circles and squares; Fig. 4B). This observation suggests that 1,25-(OH) 2 D 3 has induced sufficient de novo synthesis of PKC␤ within 24 h to render the antisense PKC␤ construct impotent. Thus, these experiments reveal that a relevant and required action of 1,25-(OH) 2 D 3 in promoting HL-60 cell differentiation is to up-regulate PKC␤ levels by increasing the synthesis of the enzyme.
Effects of PKC␤II Antisense on Cell Proliferation-Interestingly, PKC␤II antisense did not block 1,25-(OH) 2 D 3 inhibition of HL-60 cell proliferation (Fig. 6). Thus, these data show that blocking 1,25-(OH) 2 D 3 -stimulated increase in PKC␤II decreased the induction of cell differentiation by 80% but had no effect on 1,25-(OH) 2 D 3 inhibition of cell proliferation. Similar results were obtained with the less specific PKC␤ antisense construct (data not shown). DISCUSSION 1,25-(OH) 2 D 3 affects the growth and differentiation of numerous cell types (22)(23)(24)(25)(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) 2 D 3 . Expression of several genes including c-myc, c-fos, and PKC␣, PKC␤, and PKC␥ are regulated prior to the appearance of the mature monocytic-macrophage phenotype (7)(8)(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) 2 D 3 receptor (vitamin D receptor) is a substrate for PKC␤ 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) 2 D 3 's actions. Transcriptional response elements for 1,25-(OH) 2 D 3 have also been identified. Interestingly, the response element for 1,25-(OH) 2 D 3 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) 2 D 3 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) 2 D 3 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) 2 D 3 signal ultimately regulating gene expression and HL-60 cell differentiation.
Increasing evidence exists to indicate that PKC␤ plays an important role in 1,25-(OH) 2 D 3 promotion of HL-60 cell differentiation. A variant HL-60 cell line (HL-525) lacking basal levels of PKC␤ is resistant to phorbol ester-induced differentiation (18). However, susceptibility to phorbol ester differentiation was restored if HL-525 cells were transfected to overexpress PKC␤. Additionally, phorbol 12-myristate 13-acetate resistance of HL-525 cells was reversed by pretreating with 1,25-(OH) 2 D 3 , which increased PKC␤ levels (18). Also, it was shown that a 25-mer PKC␤ antisense construct different from the one used here was capable of partially blocking (averaging Ϸ30%) 1,25-(OH) 2 D 3 's induction of cell differentiation (19). Although a partial inhibition of 1,25-(OH) 2 D 3 -promoted cell differentiation was observed using their antisense construct, it had little effect on 1,25-(OH) 2 D 3 inhibition of cell proliferation. In our study, novel 15-mer PKC␤ and PKC␤II antisense constructs were found to inhibit 1,25-(OH) 2 D 3 promotion of cell differentiation by 80 -90%. However, these antisense oligonucleotides had no effect on 1,25-(OH) 2 D 3 's ability to inhibit cell proliferation. Moreover, reduction of basal levels of PKC␤ was not required for PKC␤ antisense to inhibit 1,25-(OH) 2 D 3 promotion of cell differentiation. This result suggests that blocking de novo synthesis of PKC␤ is the mechanism of action for the antisense construct. We demonstrated that PKC␤II is uniquely responsible for 1,25-(OH) 2 D 3 promotion of cell differentiation. There is controversy as to whether PKC␤I is expressed in HL-60 cells. In all reports, PKC␤I levels in unstimulated HL-60 cells is significantly lower than PKC␤II levels (19 -21, 35). In this study, we failed to detect measurable levels of PKC␤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) 2 D 3 increased PKC␤I protein levels or mRNA levels (19,35).
The findings reported here indicate that PKC␤II specifically participates in the signal transduction mechanisms employed by 1,25-(OH) 2 D 3 to promote HL-60 cell differentiation. Interestingly, we found a direct correlation between the quantitative lowering of PKC␤II protein levels and the degree of induced differentiation. The correlation between the increased levels of PKC␤ induced by 1,25-(OH) 2 D 3 and the extent of cellular differentiation (7)(8)(9) suggest that there are not spare PKC␤s in these cells. Thus, we suggest that the levels of PKC␤ 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) 2 D 3 that can be disassociated by blocking the expression of a single gene, PKC␤II. To date, several analogs of 1,25-(OH) 2 D 3 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) 2 D 3 into its capacity to promote cellular differentiation versus its capacity to inhibit cell proliferation.