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• W2070456887 abstract "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. 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 D3(1,25-(OH)2D3) 1The abbreviations used are: 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; PKC, protein kinase C. 1The abbreviations used are: 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; PKC, protein kinase C.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 (1Walters M.R. Endocr. Rev. 1992; 13: 719-764Crossref PubMed Google Scholar). It is accepted that, in part, this hormone alters cell function by enhancing or repressing expression of specific genes (2Darwish H.M. DeLuca H.F. Prog. Nucleic Acid Res. Mol. Biol. 1996; 53: 321-344Crossref PubMed Google Scholar, 3Haussler M.R. Jurutka P.W. Hsieh J.C. Thompson P.D. Selznick S.H. Haussler C.A. Whitfield G.K. Bone ( N. Y. ). 1995; 17: 33S-38SCrossref PubMed Scopus (105) Google Scholar). Other studies have revealed that 1,25-(OH)2D3 regulates cellular processes without altering gene expression (4Norman A.W. Endocr. Rev. 1982; 3: 331-336Crossref PubMed Scopus (551) Google Scholar). 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 (5Okazaki T. Bell R.M. Hannun Y.A. J. Biol. Chem. 1989; 264: 19076-19080Abstract Full Text PDF PubMed Google Scholar, 6Wali R.K. Baum C.L. Sitrin M.D. Brasitus T.A. J. Clin. Invest. 1990; 85: 1296-1303Crossref PubMed Scopus (167) Google Scholar). 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 (7Martell R.E. Simpson R.U. Taylor J.M. J. Biol. Chem. 1987; 262: 5570-5575Abstract Full Text PDF PubMed Google Scholar, 8Obeid L.M. Okazaki T. Karolak L.A. Hannun Y.A. J. Biol. Chem. 1990; 265: 2370-2374Abstract Full Text PDF PubMed Google Scholar, 9Solomon D.H O'Driscoll K. Sosne G. Weinstein I.B. Cayre Y.E. Cell Growth Differ. 1991; 2: 187-194PubMed Google Scholar). 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 (10Hug H. Sarre F.F. Biochem. J. 1993; 291: 329-343Crossref PubMed Scopus (1218) Google Scholar, 11Hoffmann J. FASEB J. 1997; 11: 649-669Crossref PubMed Scopus (334) Google Scholar). The classical PKCs, PKCα, PKCβI, PKCβII, and PKCγ, require calcium for activation. A second class of PKCs has been termed the novel PKCs and consist of PKCδ, PKCε, PKCη, and PKCθ (11Hoffmann J. FASEB J. 1997; 11: 649-669Crossref PubMed Scopus (334) Google Scholar). 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 PKCλ, PKCμ, and PKCζ. 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 (7Martell R.E. Simpson R.U. Taylor J.M. J. Biol. Chem. 1987; 262: 5570-5575Abstract Full Text PDF PubMed Google Scholar). 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 (12Martell R.E. Simpson R.U. Hsu T. Biochem. Pharmacol. 1988; 37: 635-640Crossref PubMed Scopus (27) Google Scholar, 13Simpson R.U. Hsu T. Wendt M.D. Taylor J.M. J. Biol. Chem. 1989; 264: 19710-19717Abstract Full Text PDF PubMed Google Scholar). 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 (14O'Connell, T. D., Giacherio, D. A., Jarvis, A. K., and Simpson, R. U. Endocrinology , 136, 482–488.Google Scholar, 15van Leeuwen J.P.T.M. Birkenhager J.C. van den Bemd G.-J. Buurman C.J. Staal A. Bos M.P. Pols H.A. J. Biol. Chem. 1992; 267: 12562-12569Abstract Full Text PDF PubMed Google Scholar, 16Bellito T. Boland R. Norman A.W. Boullon R. Thomasset M. Vitamin D. Gene Regulation, Structure: Functional Analysis and Clinical Application. 1991: 409-410Google Scholar, 17Weavers V.M. Franks D.J. Welsh J.E. Cell. Signalling. 1992; 4: 293-301Crossref PubMed Scopus (4) Google Scholar). 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 PKCβ is, to some extent, the isoenzyme involved in 1,25-(OH)2D3promotion of HL-60 cell differentiation (18Tonetti D.A. Henning-Chubb C. Yamanishi D.T. Huberman E. J. Biol. Chem. 1994; 269: 23230-23235Abstract Full Text PDF PubMed Google Scholar, 19Gamard C.J. Blode G.C. Hannun Y.A Obeid L.M. Cell Growth Differ. 1994; 5: 405-409PubMed Google Scholar). In this study, we showed that increased PKCβII levels by 1,25-(OH)2D3 is required to promote HL-60 cell differentiation. Interestingly, PKCβII antisense had no effect on 1,25-(OH)2D3 inhibition of HL-60 cell proliferation. Our report shows that increases in PKCβII levels and activation are important events in 1,25-(OH)2D3promotion of cell differentiation. Moreover, we suggest that the events leading to cellular differentiation most likely require protein phosphorylation. 1,25-(OH)2D3 was purchased from Tetrionics Inc. (Madison, WI). Vitamin D3metabolite purity and structural integrity were confirmed by high performance liquid chromatography and UV spectroscopy. All other reagents were reagent grade or better. 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 × 105cells/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 (7Martell R.E. Simpson R.U. Taylor J.M. J. Biol. Chem. 1987; 262: 5570-5575Abstract Full Text PDF PubMed Google Scholar, 12Martell R.E. Simpson R.U. Hsu T. Biochem. Pharmacol. 1988; 37: 635-640Crossref PubMed Scopus (27) Google Scholar, 36Pan Q. Granger J. O'Connell T.D. Somerman M.J. Simpson R.U. Biochem. Pharmacol. 1997; 54: 909-915Crossref PubMed Scopus (36) Google Scholar). Phosphorothioate oligonucleotides were synthesized by the DNA Synthesis Center at the University of Michigan. PKCβ antisense was designed to interact with bases +4 to +18 of the PKCβ mRNA. PKCβ sense had the complementary sequence of the same region. PKCβ oligonucleotides used were: PKCβ sense, 5′-GCT GAC CCG GCT GCG-3′; PKCβ antisense, 5′-CGC AGC CGG GTC AGC-3′. PKCα antisense was designed to interact with bases +6 to +20 of the PKCα mRNA. PKCα sense had the complementary sequence over the same region. PKCα oligonucleotides used were: PKCα sense, 5′-TCG GGG GGG ACC ATG-3′; PKCα antisense, 5′-CAT GGT CCC CCC CGA-3′. PKCβI and PKCβII specific antisenses were designed to interact with bases +1942 to +1956 that exist 3′ from the splice site. PKCβI sense and PKCβII sense had the complementary sequences to its respectful antisense pair. PKCβI antisense was: 5′-GTT TTA AGC ATT TCG-3′; PKCβII antisense was: 5′-GTT GGA GGT GTC TCT-3′. 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 (37Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215458) Google Scholar). 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 (PKCβ, PKCβI, PKCβII, and PKCα 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 number was determined using a Coulter (Coulter Electronics, Hialeah, FL) model Zf cell counter. Cell viability was determined using trypan blue dye exclusion. Differences between 1,25-(OH)2D3 treated cells and untreated cells for all assays were evaluated by unpaired Student's ttest. HL-60 cells were treated with control (0.1% ethanol) or 20 nm1,25-(OH)2D3 and PKCβ sense or antisense for 48 h. PKCβ protein levels were determined by Western blot analysis (Fig. 1). Cells treated with 1,25-(OH)2D3 in the presence of PKCβ sense oligonucleotide showed similar increases in PKCβ levels as compared with cells exposed to 1,25-(OH)2D3 alone. Importantly, cells treated with PKCβ antisense (lane 4) exhibited a mark inhibition in 1,25-(OH)2D3induction of PKCβ levels. PKCβ levels were decreased by 81 ± 9% (mean ± S.E.) relative to sense or oligonucleotide free cultures. The antibody used to detect PKCβ in this experiment was not specific for the splice isoenzymes βI or βII. As seen in Fig. 1, untreated (control) cells routinely exhibited minimal levels of PKCβ. Furthermore, PKCβ levels remain unchanged in uninduced (control) cells even after 48 h of PKCβ antisense treatment (n = 13, lanes 1 and 2, Fig. 1). This observation is expected, because PKCβ has a half-life of greater than 70 h. Therefore, blocking translation with PKCβ antisense would not greatly influence existing levels of PKCβ. As shown in Fig.2, PKCβ levels were increased within 24 h of 1,25-(OH)2D3 treatment. Furthermore, PKCβ antisense significantly blocked 1,25-(OH)2D3 induction of PKCβ levels at 24 and 48 h of hormone treatment. Therefore, these results demonstrate that the PKCβ antisense oligonucleotide is able to block the induction of PKCβ levels by 1,25-(OH)2D3.Figure 2Time course of PKCβ antisense treatment on 1,25-(OH)2D3-induced increases of PKCβ protein levels. HL-60 cells were treated with 20 nm1,25-(OH)2D3 for 1, 6, 24, or 48 h in the presence of 30 μm PKCβ sense or PKCβ antisense. Inlane 1, cells were treated with vehicle (Control) for 48 h. Cells were harvested at each time point, and PKCβ levels were visualized by Western blot analysis.View Large Image Figure ViewerDownload Hi-res image Download (PPT) HL-60 cells were treated with 20 nm1,25-(OH)2D3 alone (C) or with PKCα sense, PKCα antisense, PKCβ sense, or PKCβ antisense (30 μm amount of either oligonucleotide) for 48 h. PKCβ levels were determined by Western blot analysis (Fig.3 A). PKCβ levels in cells treated with 1,25-(OH)2D3 and PKCα sense, PKCα antisense, or PKCβ sense were not significantly different from cells treated with 1,25-(OH)2D3 alone. As expected, PKCβ antisense was able to inhibit the induction of PKCβ by 1,25-(OH)2D3 (lane 5). Moreover, as shown in Fig. 3 B, PKCα antisense was able to specifically block 1,25-(OH)2D3 enhancement of PKCα levels. Importantly, PKCβ antisense had no effect on 1,25-(OH)2D3 induction of PKCα levels. These results demonstrate that PKCβ antisense has specificity in blocking 1,25-(OH)2D3-induced increases of PKCβ. Western blot analysis of PKCβI and PKCβII splice isoenzymes was performed using specific antibodies. PKCβII protein levels were detectable using these specific antibodies, whereas PKCβI levels were not detectible. This observation in HL-60 cells is similar to ones reported previously (20Hocevar B.A. Fields A.P. J. Biol. Chem. 1991; 266: 28-33Abstract Full Text PDF PubMed Google Scholar, 21Tonetti D.A. Horio M. Collart F.R. Huberman E. Cell Growth Differ. 1992; 3: 739-745PubMed Google Scholar). Also, the PKCα antisense oligonucleotide at concentrations up to 60 μm had no effect on 1,25-(OH)2D3 promotion of cell differentiation (data not shown). HL-60 cells were treated with 20 nm 1,25-(OH)2D3 and either 30 μm PKCβ sense or 1, 10, or 30 μm PKCβ antisense for 48 h. PKCβ levels were determined by Western blot analysis (Fig. 4 A). As shown in Fig. 4 A, a dose-dependent decrease in PKCβ levels was observed with increasing concentrations of PKCβ antisense. In this experiment an 85% decrease, as determined by scanning densitometry, in PKCβ levels was observed with 30 μmPKCβ antisense. Next, the effects of antisense constructs on HL-60 cell differentiation and the importance of the time of PKCβ antisense addition, relative to 1,25-(OH)2D3 treatment, were also examined. HL-60 cells were treated with PKCβ sense or PKCβ antisense 24 h prior to (open symbols) or at the same time (closed symbols) as 20 nm1,25-(OH)2D3. Cell differentiation was determined by nitro blue tetrazolium dye reduction (circles) and nonspecific esterase activity (squares) (Fig.4 B). 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 PKCβ sense (data not shown). Differentiation promoted by 1,25-(OH)2D3 was inhibited by 86 ± 9% in cells pretreated with PKCβ antisense (30 μm) and 82 ± 8% in cells co-treated with PKCβ antisense (Fig. 4 B). Therefore, it is likely that the action of the antisense construct is not to lower existing PKCβ levels but to block 1,25-(OH)2D3-induced increases in PKCβ synthesis. However, if cells were first treated with 1,25-(OH)2D3 for 24 h prior to PKCβ antisense, antisense treatment was ineffective in blocking 1,25-(OH)2D3 promotion of cell differentiation (hatched circles and squares; Fig.4 B). This observation suggests that 1,25-(OH)2D3 has induced sufficient de novo synthesis of PKCβ within 24 h to render the antisense PKCβ 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 PKCβ levels by increasing the synthesis of the enzyme. HL-60 cells were treated with vehicle, 20 nm 1,25-(OH)2D3, or 20 nm 1,25-(OH)2D3 and PKCβ sense, PKCβ antisense, PKCβI sense, PKCβI antisense, PKCβII sense, or PKCβII 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. 5 A). PKCβ sense did not affect 1,25-(OH)2D3-induced expression of CD14, whereas PKCβ 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 PKCβ 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 PKCβI and PKCβII was examined. A complete block of 1,25-(OH)2D3-induced CD14 expression was observed with the PKCβII-specific antisense oligonucleotide (Fig.5 C). In contrast, PKCβI-specific antisense failed to reverse the enhanced expression of CD14 by 1,25-(OH)2D3 (Fig. 5 D). Interestingly, PKCβII 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 PKCβII decreased the induction of cell differentiation by 80% but had no effect on 1,25-(OH)2D3inhibition of cell proliferation. Similar results were obtained with the less specific PKCβ antisense construct (data not shown). 1,25-(OH)2D3 affects the growth and differentiation of numerous cell types (22Hosomi J. Hosoi J. Abe E. Suda T. Kuroki T. Endocrinology. 1983; 113: 683-689Crossref Scopus (450) Google Scholar, 23Yoneda T. Alsina M.M. Garcia J.L. Mundy G.R. Endocrinology. 1991; 129: 683-689Crossref PubMed Scopus (44) Google Scholar, 24Mangelsdorf D. Koeffler H.P. Donaldson C. Haussler M. Pike J. J. Cell Biol. 1984; 98: 391-398Crossref PubMed Scopus (400) Google Scholar, 25Simpson R.U. Hsu T. Begley D.A. Mitchell B.S. Alizadeh B.N. J. Biol. Chem. 1987; 262: 4104-4108Abstract Full Text PDF PubMed Google Scholar, 26Simpson R.U. Thomas G.A. Arnold A.J. J. Biol. Chem. 1985; 260: 8882-8891Abstract Full Text PDF PubMed Google Scholar). Relevant to this report HL-60 cells have been shown to differentiate into monocytes-macrophages (24Mangelsdorf D. Koeffler H.P. Donaldson C. Haussler M. Pike J. J. Cell Biol. 1984; 98: 391-398Crossref PubMed Scopus (400) Google Scholar) and osteoclast-like cells (23Yoneda T. Alsina M.M. Garcia J.L. Mundy G.R. Endocrinology. 1991; 129: 683-689Crossref PubMed Scopus (44) Google Scholar) upon exposure to 1,25-(OH)2D3. Expression of several genes including c-myc, c-fos, and PKCα, PKCβ, and PKCγ are regulated prior to the appearance of the mature monocytic-macrophage phenotype (7Martell R.E. Simpson R.U. Taylor J.M. J. Biol. Chem. 1987; 262: 5570-5575Abstract Full Text PDF PubMed Google Scholar, 8Obeid L.M. Okazaki T. Karolak L.A. Hannun Y.A. J. Biol. Chem. 1990; 265: 2370-2374Abstract Full Text PDF PubMed Google Scholar, 9Solomon D.H O'Driscoll K. Sosne G. Weinstein I.B. Cayre Y.E. Cell Growth Differ. 1991; 2: 187-194PubMed Google Scholar, 28Brelvi Z.S. Christakos S. Studzinski G.P. Lab. Invest. 1986; 55: 269-275PubMed Google Scholar). 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 (25Simpson R.U. Hsu T. Begley D.A. Mitchell B.S. Alizadeh B.N. J. Biol. Chem. 1987; 262: 4104-4108Abstract Full Text PDF PubMed Google Scholar, 29Reitsma P.H. Rothberg P.G. Astrin S.M. Trial J. Bar-Shavit Z. Hall A. Teitelbaum S.L. Kahn A.J. Nature. 1983; 306: 492-494Crossref PubMed Scopus (343) Google Scholar, 30Mitchell P.J. Tjian R. Science. 1989; 245: 371-378Crossref PubMed Scopus (2201) Google Scholar). 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)2D3receptor (vitamin D receptor) is a substrate for PKCβ and that phosphorylation of vitamin D receptor is important for controlling osteocalcin expression (3Haussler M.R. Jurutka P.W. Hsieh J.C. Thompson P.D. Selznick S.H. Haussler C.A. Whitfield G.K. Bone ( N. Y. ). 1995; 17: 33S-38SCrossref PubMed Scopus (105) Google Scholar, 27Desai R.K. van Wijnen A.J. Stein J.L. Stein G.S. Lian J.B. Endocrinology. 1995; 136: 5685-5693Crossref PubMed Google Scholar). 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 (31Bortell R. Owens T.A. Bidwell J.P. Gavazzo P. Breen E. van Wijnen A.J. DeLuca H.F. Stein J.L. Lian J.B. Stein G.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6119-6123Crossref PubMed Scopus (59) Google Scholar, 32MacDonald P.N. Dowd D. Nakajima S. Galligan M.A. Reeder M.C. Haussler C.A. Ozata K. Haussler M.R. Mol. Cell. Biol. 1993; 13: 5907-5917Crossref PubMed Scopus (234) Google Scholar). 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 andc-jun regulates their AP-1 binding activity (33Bohmann D. Tjian R. Cell. 1989; 59: 709-717Abstract Full Text PDF PubMed Scopus (145) Google Scholar). 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 (34Martell R.E. Strahler J.R. Simpson R.U. J. Biol. Chem. 1992; 267: 7511-7518Abstract Full Text PDF PubMed Google Scholar). Our laboratory reported that 10 nuclear proteins undergo phosphorylation state changes within 6–40 h of 1,25-(OH)2D3 treatment (34Martell R.E. Strahler J.R. Simpson R.U. J. Biol. Chem. 1992; 267: 7511-7518Abstract Full Text PDF PubMed Google Scholar). 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 PKCβ 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 PKCβ is resistant to phorbol ester-induced differentiation (18Tonetti D.A. Henning-Chubb C. Yamanishi D.T. Huberman E. J. Biol. Chem. 1994; 269: 23230-23235Abstract Full Text PDF PubMed Google Scholar). However, susceptibility to phorbol ester differentiation was restored if HL-525 cells were transfected to overexpress PKCβ. Additionally, phorbol 12-myristate 13-acetate resistance of HL-525 cells was reversed by pretreating with 1,25-(OH)2D3, which increased PKCβ levels (18Tonetti D.A. Henning-Chubb C. Yamanishi D.T. Huberman E. J. Biol. Chem. 1994; 269: 23230-23235Abstract Full Text PDF PubMed Google Scholar). Also, it was shown that a 25-mer PKCβ antisense construct different from the one used here was capable of partially blocking (averaging ≈30%) 1,25-(OH)2D3‘s induction of cell differentiation (19Gamard C.J. Blode G.C. Hannun Y.A Obeid L.M. Cell Growth Differ. 1994; 5: 405-409PubMed Google Scholar). 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 PKCβ and PKCβII 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 PKCβ was not required for PKCβ antisense to inhibit 1,25-(OH)2D3 promotion of cell differentiation. This result suggests that blocking de novo synthesis of PKCβ is the mechanism of action for the antisense construct. We demonstrated that PKCβII is uniquely responsible for 1,25-(OH)2D3 promotion of cell differentiation. There is controversy as to whether PKCβI is expressed in HL-60 cells. In all reports, PKCβI levels in unstimulated HL-60 cells is significantly lower than PKCβII levels (19Gamard C.J. Blode G.C. Hannun Y.A Obeid L.M. Cell Growth Differ. 1994; 5: 405-409PubMed Google Scholar, 20Hocevar B.A. Fields A.P. J. Biol. Chem. 1991; 266: 28-33Abstract Full Text PDF PubMed Google Scholar, 21Tonetti D.A. Horio M. Collart F.R. Huberman E. Cell Growth Differ. 1992; 3: 739-745PubMed Google Scholar, 35Devalia D. Thomas S.B. Roberts P.J. Jones H.M. Linch D.C. Blood. 1992; 80: 68-76Crossref PubMed Google Scholar). In this study, we failed to detect measurable levels of PKCβI. This finding is in agreement with several reports (20Hocevar B.A. Fields A.P. J. Biol. Chem. 1991; 266: 28-33Abstract Full Text PDF PubMed Google Scholar, 21Tonetti D.A. Horio M. Collart F.R. Huberman E. Cell Growth Differ. 1992; 3: 739-745PubMed Google Scholar). However, others have shown, using different antibodies or Northern blot analysis, that 1,25-(OH)2D3 increased PKCβI protein levels or mRNA levels (19Gamard C.J. Blode G.C. Hannun Y.A Obeid L.M. Cell Growth Differ. 1994; 5: 405-409PubMed Google Scholar, 35Devalia D. Thomas S.B. Roberts P.J. Jones H.M. Linch D.C. Blood. 1992; 80: 68-76Crossref PubMed Google Scholar). The findings reported here indicate that PKCβII 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 PKCβII protein levels and the degree of induced differentiation. The correlation between the increased levels of PKCβ induced by 1,25-(OH)2D3 and the extent of cellular differentiation (7Martell R.E. Simpson R.U. Taylor J.M. J. Biol. Chem. 1987; 262: 5570-5575Abstract Full Text PDF PubMed Google Scholar, 8Obeid L.M. Okazaki T. Karolak L.A. Hannun Y.A. J. Biol. Chem. 1990; 265: 2370-2374Abstract Full Text PDF PubMed Google Scholar, 9Solomon D.H O'Driscoll K. Sosne G. Weinstein I.B. Cayre Y.E. Cell Growth Differ. 1991; 2: 187-194PubMed Google Scholar) suggest that there are not spare PKCβs in these cells. Thus, we suggest that the levels of PKCβ 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, PKCβII. 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. We thank Li Huang for her technical efforts and expertise." @default.
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