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Protein Kinase Cα Is an Effector of Hexamethylene Bisacetamide-Induced Differentiation of Friend Erythroleukemia Cells
Experimental Cell Research 246, 348 –354 (1999) Article ID excr.1998.4312, available online at http://www.idealibrary.com on
Protein Kinase Ca Is an Effector of Hexamethylene BisacetamideInduced Differentiation of Friend Erythroleukemia Cells Conrad M. Mallia, Victoria Aguirre, Eric McGary, Yan Tang, Aline B. Scandurro, Chun Liu, Constance T. Noguchi, and Barbara S. Beckman 1 Department of Pharmacology, Tulane Cancer Center, Tulane University School of Medicine, New Orleans, Louisiana 70112; and Laboratory of Chemical Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892
The program of biochemical and molecular events necessary for commitment to erythroid cell differentiation is particularly well characterized in murine Friend erythroleukemia cell lines. Commitment to hemoglobin synthesis in response to a variety of chemical inducers, including hexamethylene bisacetamide and dimethyl sulfoxide is completed by 24 h and proceeds to terminal differentiation by 96 h. Phorbol 12myristate 13-acetate, a classical tumor promoter phorbol ester that binds to protein kinase C, blocks differentiation in a reversible manner, suggesting an important role for protein kinase C signaling pathways. The classical protein kinase C isoforms a, bI, and bII, play distinct roles in the transduction of proliferative and differentiative signals in human, as well as in murine, erythroleukemia cells. Protein kinase Ca has been implicated in differentiation of human erythroleukemia cells although its translocation to the nucleus has not been observed. Taking advantage of the ability of phorbol 12-myristate 13-acetate to block differentiation in Friend erythroleukemia cells, we determined the localization of the predominant protein kinase C isoforms a and bI during differentiation and in response to their blockade. The ability of phorbol myristate acetate to preferentially diminish protein kinase Ca-protein localization to the nucleus by 24 h and thereby block differentiation induced by hexamethylene bisacetamide was paralleled by the ability of protein kinase Ca antisense transfection to block differentiation. In addition, b-globin transcription, assessed by polymerase chain reaction, was significantly decreased in protein kinase Ca antisense-transfected cells compared to that seen in vector transfected ones. Taken together, these data suggest an important temporal role for nuclear protein kinase Ca localization in Friend erythroleukemia cell differentiation. © 1999 Academic Press
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To whom reprint requests should be addressed at Department of Pharmacology SL83, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA 70112. Fax: (504) 588-5283. 0014-4827/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
Key Words: protein kinase Ca; b-globin; erythroleukemia.
INTRODUCTION
Long before phorbol esters were known to activate the family of serine/threonine kinases called protein kinase C (PKC), their role in the promotion of skin cancer in animal models had been well studied. The ability of a single dose of phorbol 12-myristate 13acetate (PMA) to induce skin cancer in an appropriately pretreated mouse and also to completely downregulate PKC in a long-term fashion (3– 4 days) implicated PKC in this process [1]. It is now clear that PKC is an essential mediator of cell signaling [2], particularly in hematopoietic cells [3], although specific details of how this family of enzymes mediates its integral role in signal transduction cascades are lacking. The best evidence for distinct roles of PKC isoforms in these processes has been obtained in hematopoietic cell lines, such as the human promyelocytic leukemia cell HL-60, that differentiate into macrophages in response to PMA and into monocytes in response to 1,25-dihydroxy vitamin D 3 [4, 5]. Antisense technology helped to define distinct roles for PKCa and PKCb in differentiation and proliferation. In the K562 human erythroleukemia cell line, PKCbII has been strongly implicated in proliferative events via lamin B phosphorylation at the nuclear envelope, whereas PKCa appeared to be critical for megakaryocyte differentiation [6]. Murine Friend erythroleukemia cell (FELC) lines provide another well-studied model of programmed cell differentiation. Various chemicals, such as hexamethylene bisacetamide (HMBA) [7] and dimethyl sulfoxide (DMSO) [8], induce synthesis of hemoglobin and promote the arrest of cell growth. These and other changes appear to resemble those occurring at some stages of normal erythroid differentiation. The diacetylated diamine HMBA is the most effective inducer of erythroid differentiation in FELC to date, yet its mechanism of
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action is still not clear. The most plausible hypothesis is that inducers act by affecting the function of the plasma membrane [9]. Diacylglycerol levels, in particular, have been correlated with FELC differentiation [10] or its inhibition by phorbol esters [8]. The classical PKC isoforms (a, bI, bII, and g), as well as the novel ones (d, e, h, and u), are receptors for diacylglycerol. The calcium-dependent isoforms PKCa and b [11–13], as well as the calcium-independent PKC isoforms d and e [14], have been linked to differentiation in FELC. Phorbol ester tumor promoters, such as phorbol 12myristate 13-acetate (PMA), effectively inhibit the terminal differentiation of FELC. The observation by Thomas et al. [15] that phorbol ester treatment of NIH 3T3 cells resulted in translocation of PKCa to the nucleus, as well as the correlation of proliferative events with PKCb isoforms in the nuclei of hematopoietic cell lines [11, 16], prompted this examination of the temporal pattern of PKCa and bI isoform localization during HMBA-induced differentiation and during its blockade by PMA. We demonstrate in this report that PMA treatment of FELC affects the time-course of nuclear PKCa isoform localization in response to HMBA. HMBA-induced differentiation, as measured by b-globin expression, was also significantly blocked in FELC expressing PKCa antisense. These results suggest that the appropriate temporal PKCa-protein localization to the plasma membrane and to the nucleus is essential for HMBA-induced erythroid cell differentiation. MATERIALS AND METHODS Cell culture. The GM86 murine erythroleukemia cell line (clone of the Friend erythroleukemia cell line) was obtained from the NIGMS Human Genetic Mutant Cell Repository. It was maintained in log-phase growth by passage twice weekly in enriched MEM (GibcoBRL, Grand Island, NY) supplemented with 10% fetal bovine serum (BioWhittaker), 1% penicillin–streptomycin (GibcoBRL), 2% nonessential amino acids (GibcoBRL), 1% L-glutamine (GibcoBRL), 1% fungizone (GibcoBRL), and 2% MEM vitamins (GibcoBRL). Cells were incubated at 37°C in humidified air with 5% CO 2. Differentiation studies. Log-phase cultures were treated with HMBA (5 mM), PMA (100 ng/ml), or both HMBA and PMA (Sigma Chemical Co.). Cultures were incubated for 96 h. Differentiation was evaluated by determining the percentage of benzidine-positive cells [8]. FELC lysate preparation. GM86 FELC were collected at different experimental times (0, 0.5, 6, 24, and 96 h). Following washing twice with ice-cold PBS and centrifugation (1000g for 5 min), pelleted cells were lysed by sonicating for 30 s in a buffer containing 62.5 mM phenylmethylsulfonlyl fluoride (PMSF), 25 mg/ml leupeptin, and 25 mg/ml aprotinin. After centrifugation (100g for 20 min), the resulting supernatants were collected and their protein concentrations were measured by the method of Bradford [17]. Subcellular FELC fractionation. Cytosol and membrane FELC fractionations were performed according to an adapted procedure from Lehel et al. [18]. Briefly, after counting and washing cells, the resulting pellets were treated with lysis buffer (20 mM Tris–HCl, 2 mM EGTA, pH 7.4, with protease inhibitors) (10 mL/L 3 10 6 cells).
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Suspensions were passed through 25-gauge syringes 10 times in an ice bath and spun at 1000g for 10 min at 4°C. Supernatants were centrifuged twice (2000g for 5 min and 30,000g for 30 min at 4°C). These last supernatants were saved as cytosolic fractions and pellets saved as membrane fractions. Protein kinase C was extracted from the membrane fraction pellets by the method of Leach et al. [19]. Lysis buffer with 1% Triton was added to the pellets (3 mL/L 3 10 6 cells). Tubes were vortexed and sonicated for 10 s. This lysate was then considered to be the membrane extract. Isolation of nuclei. Nuclei were isolated according to the method of Neumann et al. [20] and were free of cytoplasmic contamination, as determined by phase-contrast microscopy. Cells were counted, washed with cold PBS one time, and centrifuged at 4°C for 5 min at 800g. Pellets were washed once with 10 ml cold sucrose–TKM buffer (0.25 M sucrose, 0.025 M KCl, 0.005 M MgCl 2, 0.05 M Tris–HCl, pH 7.5) and spun for 10 min at 1000g at 4°C. Pellets were resuspended at 5–20 3 10 6 cells/ml in 10 ml sucrose TKM buffer containing 0.1% Triton X-100 and 0.05 M PMSF. Cells were disrupted in a Dounce homogenizer with about 10 strokes of a loose-fitting pestle on ice. FELC were then centrifuged at 1000g for 10 min at 4°C to harvest nuclei. Pellets were saved and resuspended in 10 ml of cold sucrose TKM to wash nuclei. After spinning for 10 min at 1000g at 4°C, pellets were stored frozen with protein inhibitors. Nuclear protein extraction. Frozen pellets were thawed and resuspended in 0.5 ml high-salt buffer (0.5 M NaCl, 0.05 M MgCl 2, 0.01 M Tris–HCl, pH 7.5, 10 mL/mL PMSF, 25 mL/mL DNase, 10 mL/mL of aprotinin, and 20 mL/mL of leupeptin) in a proportion of 2–3 3 10 7 nuclei/ml. Tubes were incubated at 37°C for 20 min, chilled on ice to stop reactions, and centrifuged at 12,000g (2300 rpm) for 10 min at 4°C. Supernatants were reserved as nuclear protein isolates. Immunodetection of PKC isoforms. Proteins (20 –100 mg) from cell fractions were electrophoretically separated on a 0.1% sodium dodecyl sulfate (SDS)–7.5% polyacrylamide slab minigel, and membrane proteins (50 mg) were fractionated with 6% SDS–PAGE, according to the method of Laemmli [21]. Proteins were transferred to a nitrocellulose membrane using an adapted technique from the original Western blot procedure [22]. Membranes were blocked with 5% Carnation low-fat milk in PBS containing 0.05% Tween-20 and incubated with rabbit polyclonal antibodies to isoform-specific peptides of the a, bI, bII, g, d, e, h, and z PKC (Oxford Biomedical Research Inc., Oxford, MI; R & D Antibodies, Berkeley, CA) or obtained from Alan Fields, University of Texas (Galveston) for 2 h at room temperature. Membranes were washed three times for 10 min with PBS–Tween solution, and then they were incubated 30 min with goat anti-rabbit (GAR) antibody conjugated to horseradish peroxidase (HDR) diluted 1:30,000 (Kirkegard and Perry, Gaithersburg, MD). After three brief washes with PBS–Tween solution, antigen– antibody complexes were detected using enhanced chemiluminescence (Amersham Life Science, Inc., Arlington Heights, IL) on autoradiographic film. Baculovirus expressed specific PKC isoforms (Oxford) were used as positive controls for the PKC antibodies. Antibodies have been characterized by their manufacturers to be PKC isoform specific. The molecular weights of bands were determined using protein molecular weight standards (Sigma Chemical Co., St. Louis, MO) that were electrophoretically separated and transferred at the same time as the protein samples. Protein blots were stained with 0.5% Ponceau S solution to confirm equal loading of the lanes. Transfection of GM86 cells with PKCa antisense. Human PKCa (obtained from American Type Culture Collection, Rockville, MD) was subcloned into the EcoRI site of pBluescript KS 1 (Stratagene, LaJolla, CA). (The homology between human and murine PKCa is 89%.) Digestion with HindIII was followed by further ligation and the selection of transformants with the PKCa cDNA in the antisense orientation. Lipofectin was used for transfections according to manufacturer’s instructions. Stable transfectants were selected in G418. PKCa protein expression was verified by Western blot analysis.
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cause PMA had no significant effect on PKCd and e, these isoforms were excluded from further consideration.
FIG. 1. Cytosolic and nuclear distribution of the a, bI, and bII isoforms of PKC. FELC were separated into their cytosolic and nuclear fractions, run on a 7.5% polyacrylamide gel, and then transferred to a nitrocellulose membrane. 5 3 106 cells were used for each of the samples. The samples were then probed with PKC isoformspecific antibodies for the indicated isoforms of PKC. N indicates the lanes containing the nuclear fractions, while C indicates the lanes containing the cytosolic fractions.
Untransfected or transfected (vector only or PKCa antisense) FELC were treated with 5 mM HMBA or 100 ng/ml PMA. Cultures were incubated for 96 h. Differentiation was evaluated by determining the percentage of benzidine positive cells [8]. RNA extraction and PCR determination of b-globin. For quantification of RNA using competitive PCR, 1 mg total RNA was reversetranscribed in a 20-ml reaction mixture using reverse transcriptase (Perkin-Elmer, Foster City, CA). The product was then subjected to competitive PCR to measure the b-globin transcription level using primers 59-GCTGAGAAGGCTGCTGTCT-39 (sense) and 59-ACCTTCTGGAAGGCAGCC-39 (antisense). As an internal control, RNA for the ubiquitous ribosomal protein S16 was also quantitated using 59-CTGGAGCCTGTTTTGTTCTG-39 (sense) and 59-TGAGATGGACTGTCGGATGG-39 (antisense). In a typical quantitation, a series of reactions was set up with increasing amounts of sample and concomitant decreasing amounts of standard (prepared as described below) at a twofold interval. The resulting products were analyzed on a 2% agarose gel and scanned using a laser densitometer (Molecular Dynamics, Los Alamos, CA). The condition at which the sample band and the standard band display similar intensities was used to determine the amount of corresponding cDNA in the PCR reaction [23]. Standard for competitive RT-PCR of the mouse b-globin was prepared from plasmid containing the mouse b-globin cDNA by PCR with primers 59-GCTGAGAAGGCTGCTGTCTACCCTTGGACCCAGCGGTACTTTGAT-39 (sense) and 59-ACCTTCTGGAAGGCAGCC-39 (antisense). The product of such a reaction serves as template for the original pair of b-globin primers yielding a shorter product (311 bp) distinguishable from the natural product (380 bp). Standard for the ribosomal protein S16 was generated similarly. The PCR products were gel purified, electroeluted, and the concentration determined by UV absorbance at 260 nm. The product was then coamplified in the quantitative RT-PCR reactions, as described above.
HMBA and PMA Induced Changes in PKC Isoform Expression Results shown in Table 1 confirm previous findings that PMA inhibits HMBA-induced differentiation in Friend erythroleukemia cells [7]. The localization of PKCa and bI to the nuclear, membrane, or cytosolic compartment was examined in response to HMBA or PMA treatment alone or in combination at 0, 0.5, 24, or 96 h. Figure 2A shows that nuclear localization of PKCa was maximal after 24 h of HMBA treatment, whereas nuclear PKC bI decreased throughout the treatment period (Fig. 2B). PKC bII was not further studied because of its weaker nuclear signal on immunoblot. With PMA treatment, the pattern of protein localization changed. Nuclear PKCa protein levels peaked at 0.5 h during PMA or combination treatment and were no longer observed at 24 or 96 h. Nuclear PKCbI protein levels decreased at 0.5 h and returned to control levels by 24 h. With combination treatment, PKCbI protein levels decreased at 0.5 h and were absent at 24 h, followed by a reappearance of protein expression at 96 h. Both cytosolic and membrane fractions of FELC subjected to the same treatments over time exhibited a downregulation of PKCa by 24 h that was maintained at 96 h (Figs. 3A and 3B). PKCa protein levels were elevated at the same time points in HMBA-treated cells. The combination of PMA and HMBA treatment resulted in total downregulation of PKCa at 96 h in the cytosolic, membrane, and nuclear compartments, suggesting that the ability of PMA to block HMBA-induced differentiation might be the result of a global downregulation in PKCa. HMBA-Induced Differentiation and Its Inhibition by PMA and PKCa Antisense Because PKCa protein localization to the nucleus occurred within the 24-h period of commitment, and
RESULTS
Cellular Distribution of PKC Isoforms in FELC The cytosol and nuclear fractions of Friend erythroleukemia cells in the logarithmic phase of growth were isolated and probed for PKC isoform expression. Although the a isoform was the most abundant, it was localized mainly in the cytosol fraction of the cells. The bI and bII isoforms, on the other hand, were found in the nucleus (Fig. 1), as well as cytosol. The novel PKC isoforms d and e were also examined. PKCe was only faintly detected in the cytosol and did not localize to the nucleus, and PKCd was absent from the cytosol and faintly detected in the nucleus (data not shown). Be-
TABLE 1 Differentiation in FELC Treated with HMBA Alone, PMA Alone, or a Combination of the Two Treatment
% Differentiation
Control HMBA PMA HMBA 1 PMA
0 6 2a 70 6 5 162 562
Note. Cells were treated for 96 h with HMBA (5 mM), PMA (100 ng/ml), or both. The percentage of benzidine cells was determined for four experiments. a Mean 6 SEM.
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FIG. 2. Expression of PKCa or PKCbI in the nucleus after treatment with 5 mM HMBA, 100 ng/ml PMA, or 5 mM HMBA 1 100 ng/ml PMA. FELC were treated with the chemicals for 0, 0.5, 24, or 96 h, and the nuclear fractions were then probed for the a isoform (A) or the bI isoform (B). Immunoblots are representative of three separate experiments.
because PMA downregulated PKCa protein levels by 24 h, we tested the effect of transfecting FELC with antisense PKCa or with vector alone. As shown in Fig. 4, transfection of FELC with PKCa antisense also inhibited HMBA-induced differentiation by 41%, as did PMA compared to untransfected cells or vector control cells (not shown). Inhibition of b-Globin Gene Expression with PKCa Antisense Transfection Because b-globin is the principal gene expressed during erythroid differentiation, we tested the hypothesis that PKCa-mediated inhibition of differentiation might affect the level of b-globin expression. As shown in Fig. 5, an 85% reduction of b-globin mRNA expression in PKCa antisense expressing cells was measured after 72 h of HMBA treatment, compared to cells transfected with vector alone. A 40% reduction, compared to vector control of b-globin mRNA, was seen even after 96 h of treatment. DISCUSSION
Depending on the cell type studied, phorbol esters can either induce differentiation or block terminal differentiation. Specific isoforms of PKC have been impli-
cated in these processes. The clearest association of PKC isoforms with proliferation and differentiation has been documented for human leukemia cell lines [2, 5, 6, 24, 25]. Calcium-dependent PKCa and PKCb have been associated with differentiation and proliferation, respectively, in human K562 erythroleukemia cells [6, 25]. Only PKCbII translocated to the nucleus in response to proliferative signals in K562 cells. Antisense PKCbII blocked proliferation. PMA-induced cytostasis was reversed upon removal of PMA, and resumption of proliferation was accompanied by reexpression of PKCbII to near control levels, whereas PKCa and z levels remained elevated for several days after removal of PMA. PMA treatment of human erythroleukemia cells causes megakaryocytic differentiation and cessation of proliferation, whereas sodium butyrate induces megakaryocytic differentiation without cytostasis and causes increases in both PKCa and PKCbII in whole cell lysates. We have found that a murine erythroleukemia cell line contains the a, bI, bII, d, and e isoforms of PKC. Each of these PKC isoforms has been implicated to some extent in proliferation/differentiation events of FELC. Most recently, we have shown that PKCb binds to DNA in FELC and is closely associated with proliferative events in these cells [26]. Several additional
FIG. 3. Effect of HMBA and PMA on cytosolic and membrane localization of PKCa. FELC were treated with 100 ng/ml PMA, 5 mM HMBA, or both for the time indicated. (A) Immunoblot of cytosolic fraction or (B) membrane fraction. Representative of three experiments.
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FIG. 4. HMBA-induced differentiation of FELC. Wild type FELC were either treated with 5 mM HMBA (bar labeled Untransfected) to induce differentiation or 5 mM HMBA 1 100 ng/ml PMA (bar labeled PMA) to inhibit the HMBA effect. HMBA (5 mM) was added to “vector only” transfected cells or cells that were transfected with the antisense cDNA to PKCa (bar labeled Antisense). Differentiated cells were scored after 96 h of incubation by benzidine staining. Mean (6 SE) values: 78.9 6 1.3% for wild type cells treated with HMBA only (versus 72 6 1.5% for vector transfected cells), 1.5 6 0.2% for cells that were treated additionally with PMA, and 33 6 2.1% for PKCa antisense transfected cells (n 5 3).
studies have demonstrated that PKC activation is important early in HMBA-induced differentiation followed by PKC downregulation [7, 11, 13–14]. Leng et al. [14] found that translocation of the d and e PKC isoforms from the soluble portion of the cell to the particulate portion occurred after 15–30 min of stimulation with HMBA. Although we confirmed a translocation of the d and e PKC isoforms from the soluble to particulate fraction of FELC within 30 min of stimulation with HMBA, we found no nuclear localization of these isoforms in response to HMBA treatment. In contrast, the localization of the predominant PKCa isoform to the nucleus in a time-dependent manner coincident with the commitment phase of differentiation was pursued in terms of the effect of phorbol ester to block this HMBA-induced differentiation event. Changes in nuclear PKCbI were observed but because constitutive expression was seen in untreated cells, changes were not pursued in terms of correlations with the differentiation program. Although nuclear PKC protein activity and expression have been correlated with cell differentiation in
other cell types [24, 27, 30], very few studies have addressed the temporal relationship between PKCa isoform localization to the nucleus and commitment to differentiation. The ability of phorbol ester to reverse HMBA-induced differentiation of FELC has never been adequately explained. Although changes in diacylglycerol and PKC have been correlated with the inhibition of differentiation, the role of specific PKC isoform localized to the nucleus had not been previously addressed. Because cell lines and their variants differ in their PKC isoform distribution and their ability to differentiate in response to phorbol esters, it will be necessary to study a number of model systems before a clear picture of the molecular details of signaling pathways critical for these events can be ascertained. In the GM-86 clone of FELC, which is a growth factor independent, constitutively proliferating cell line, the cytosol and nuclear distribution of various PKC isoforms indicates that the more abundant PKCa isoform is primarily located in the cytosol, suggesting inactivation, while the bI and bII isoforms are primarily in the nucleus. The nature of their temporal distribution in response to the induction of differentiation and its inhibition provides support for the hypothesis that PKCa is critical for erythroid cell differentiation and PKCb isoforms are important for erythroid cell proliferation [6, 25, 26, 28]. The isoforms critical for regulating these biological events are likely to localize to the appropriate compartments in the cell where essential substrates are located. Our focus on the nuclear compartmentalization of PKC isoforms has been influenced by our findings, as well as others, that PKCb isoforms are
FIG. 5. b-Globin RNA expression during HMBA-induced differentiation. HMBA (5 mM) was used to induce differentiation in wild type (WT, short dashed line), vector transfected (V, long dashed line), or PKCa antisense transfected (A, solid line) cells. b-Globin mRNA expression was measured at 24-, 48-, 72-, and 96-h time points. b-Globin mRNA expression was measured using RT-PCR and is expressed as a ratio, compared to S16 mRNA.
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localized to the nucleus in response to erythroid cellspecific growth factors, such as erythropoietin, IL-3, and GM-CSF [31–32]. Although activity levels of PKC were not assessed in the current study, earlier studies from our laboratory have reported nuclear PKC activity in FELC treated with HMBA or PMA [28]. The greatest activity in the nucleus was seen in logarithmically growing cells, particularly those treated with PMA. The finding that PKCa localization to the nucleus is an early event in erythroid cell differentiation and is critical for b-globin expression supports the hypothesis that PKCa is an important component of the signal transduction pathway to gene expression. The ability of PKCa antisense transfection of FELC to block HMBA-induced differentiation, as did PMA, further supports an important role for PKCa in differentiation. REFERENCES 1.
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Mallia, C. M., Smith, M., Clejan, S., and Beckman, B. S. (1998). Erythropoietin stimulates nuclear localization of diacylglycerol and protein kinase C bII in B6SUt.EP cells. J. Lipid Med. Cell Signal. 17, 135–150.
Protein Kinase Ca Is an Effector of Hexamethylene BisacetamideInduced Differentiation of Friend Erythroleukemia Cells Conrad M. Mallia, Victoria Aguirre, Eric McGary, Yan Tang, Aline B. Scandurro, Chun Liu, Constance T. Noguchi, and Barbara S. Beckman 1 Department of Pharmacology, Tulane Cancer Center, Tulane University School of Medicine, New Orleans, Louisiana 70112; and Laboratory of Chemical Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892
The program of biochemical and molecular events necessary for commitment to erythroid cell differentiation is particularly well characterized in murine Friend erythroleukemia cell lines. Commitment to hemoglobin synthesis in response to a variety of chemical inducers, including hexamethylene bisacetamide and dimethyl sulfoxide is completed by 24 h and proceeds to terminal differentiation by 96 h. Phorbol 12myristate 13-acetate, a classical tumor promoter phorbol ester that binds to protein kinase C, blocks differentiation in a reversible manner, suggesting an important role for protein kinase C signaling pathways. The classical protein kinase C isoforms a, bI, and bII, play distinct roles in the transduction of proliferative and differentiative signals in human, as well as in murine, erythroleukemia cells. Protein kinase Ca has been implicated in differentiation of human erythroleukemia cells although its translocation to the nucleus has not been observed. Taking advantage of the ability of phorbol 12-myristate 13-acetate to block differentiation in Friend erythroleukemia cells, we determined the localization of the predominant protein kinase C isoforms a and bI during differentiation and in response to their blockade. The ability of phorbol myristate acetate to preferentially diminish protein kinase Ca-protein localization to the nucleus by 24 h and thereby block differentiation induced by hexamethylene bisacetamide was paralleled by the ability of protein kinase Ca antisense transfection to block differentiation. In addition, b-globin transcription, assessed by polymerase chain reaction, was significantly decreased in protein kinase Ca antisense-transfected cells compared to that seen in vector transfected ones. Taken together, these data suggest an important temporal role for nuclear protein kinase Ca localization in Friend erythroleukemia cell differentiation. © 1999 Academic Press
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To whom reprint requests should be addressed at Department of Pharmacology SL83, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA 70112. Fax: (504) 588-5283. 0014-4827/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
Key Words: protein kinase Ca; b-globin; erythroleukemia.
INTRODUCTION
Long before phorbol esters were known to activate the family of serine/threonine kinases called protein kinase C (PKC), their role in the promotion of skin cancer in animal models had been well studied. The ability of a single dose of phorbol 12-myristate 13acetate (PMA) to induce skin cancer in an appropriately pretreated mouse and also to completely downregulate PKC in a long-term fashion (3– 4 days) implicated PKC in this process [1]. It is now clear that PKC is an essential mediator of cell signaling [2], particularly in hematopoietic cells [3], although specific details of how this family of enzymes mediates its integral role in signal transduction cascades are lacking. The best evidence for distinct roles of PKC isoforms in these processes has been obtained in hematopoietic cell lines, such as the human promyelocytic leukemia cell HL-60, that differentiate into macrophages in response to PMA and into monocytes in response to 1,25-dihydroxy vitamin D 3 [4, 5]. Antisense technology helped to define distinct roles for PKCa and PKCb in differentiation and proliferation. In the K562 human erythroleukemia cell line, PKCbII has been strongly implicated in proliferative events via lamin B phosphorylation at the nuclear envelope, whereas PKCa appeared to be critical for megakaryocyte differentiation [6]. Murine Friend erythroleukemia cell (FELC) lines provide another well-studied model of programmed cell differentiation. Various chemicals, such as hexamethylene bisacetamide (HMBA) [7] and dimethyl sulfoxide (DMSO) [8], induce synthesis of hemoglobin and promote the arrest of cell growth. These and other changes appear to resemble those occurring at some stages of normal erythroid differentiation. The diacetylated diamine HMBA is the most effective inducer of erythroid differentiation in FELC to date, yet its mechanism of
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action is still not clear. The most plausible hypothesis is that inducers act by affecting the function of the plasma membrane [9]. Diacylglycerol levels, in particular, have been correlated with FELC differentiation [10] or its inhibition by phorbol esters [8]. The classical PKC isoforms (a, bI, bII, and g), as well as the novel ones (d, e, h, and u), are receptors for diacylglycerol. The calcium-dependent isoforms PKCa and b [11–13], as well as the calcium-independent PKC isoforms d and e [14], have been linked to differentiation in FELC. Phorbol ester tumor promoters, such as phorbol 12myristate 13-acetate (PMA), effectively inhibit the terminal differentiation of FELC. The observation by Thomas et al. [15] that phorbol ester treatment of NIH 3T3 cells resulted in translocation of PKCa to the nucleus, as well as the correlation of proliferative events with PKCb isoforms in the nuclei of hematopoietic cell lines [11, 16], prompted this examination of the temporal pattern of PKCa and bI isoform localization during HMBA-induced differentiation and during its blockade by PMA. We demonstrate in this report that PMA treatment of FELC affects the time-course of nuclear PKCa isoform localization in response to HMBA. HMBA-induced differentiation, as measured by b-globin expression, was also significantly blocked in FELC expressing PKCa antisense. These results suggest that the appropriate temporal PKCa-protein localization to the plasma membrane and to the nucleus is essential for HMBA-induced erythroid cell differentiation. MATERIALS AND METHODS Cell culture. The GM86 murine erythroleukemia cell line (clone of the Friend erythroleukemia cell line) was obtained from the NIGMS Human Genetic Mutant Cell Repository. It was maintained in log-phase growth by passage twice weekly in enriched MEM (GibcoBRL, Grand Island, NY) supplemented with 10% fetal bovine serum (BioWhittaker), 1% penicillin–streptomycin (GibcoBRL), 2% nonessential amino acids (GibcoBRL), 1% L-glutamine (GibcoBRL), 1% fungizone (GibcoBRL), and 2% MEM vitamins (GibcoBRL). Cells were incubated at 37°C in humidified air with 5% CO 2. Differentiation studies. Log-phase cultures were treated with HMBA (5 mM), PMA (100 ng/ml), or both HMBA and PMA (Sigma Chemical Co.). Cultures were incubated for 96 h. Differentiation was evaluated by determining the percentage of benzidine-positive cells [8]. FELC lysate preparation. GM86 FELC were collected at different experimental times (0, 0.5, 6, 24, and 96 h). Following washing twice with ice-cold PBS and centrifugation (1000g for 5 min), pelleted cells were lysed by sonicating for 30 s in a buffer containing 62.5 mM phenylmethylsulfonlyl fluoride (PMSF), 25 mg/ml leupeptin, and 25 mg/ml aprotinin. After centrifugation (100g for 20 min), the resulting supernatants were collected and their protein concentrations were measured by the method of Bradford [17]. Subcellular FELC fractionation. Cytosol and membrane FELC fractionations were performed according to an adapted procedure from Lehel et al. [18]. Briefly, after counting and washing cells, the resulting pellets were treated with lysis buffer (20 mM Tris–HCl, 2 mM EGTA, pH 7.4, with protease inhibitors) (10 mL/L 3 10 6 cells).
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Suspensions were passed through 25-gauge syringes 10 times in an ice bath and spun at 1000g for 10 min at 4°C. Supernatants were centrifuged twice (2000g for 5 min and 30,000g for 30 min at 4°C). These last supernatants were saved as cytosolic fractions and pellets saved as membrane fractions. Protein kinase C was extracted from the membrane fraction pellets by the method of Leach et al. [19]. Lysis buffer with 1% Triton was added to the pellets (3 mL/L 3 10 6 cells). Tubes were vortexed and sonicated for 10 s. This lysate was then considered to be the membrane extract. Isolation of nuclei. Nuclei were isolated according to the method of Neumann et al. [20] and were free of cytoplasmic contamination, as determined by phase-contrast microscopy. Cells were counted, washed with cold PBS one time, and centrifuged at 4°C for 5 min at 800g. Pellets were washed once with 10 ml cold sucrose–TKM buffer (0.25 M sucrose, 0.025 M KCl, 0.005 M MgCl 2, 0.05 M Tris–HCl, pH 7.5) and spun for 10 min at 1000g at 4°C. Pellets were resuspended at 5–20 3 10 6 cells/ml in 10 ml sucrose TKM buffer containing 0.1% Triton X-100 and 0.05 M PMSF. Cells were disrupted in a Dounce homogenizer with about 10 strokes of a loose-fitting pestle on ice. FELC were then centrifuged at 1000g for 10 min at 4°C to harvest nuclei. Pellets were saved and resuspended in 10 ml of cold sucrose TKM to wash nuclei. After spinning for 10 min at 1000g at 4°C, pellets were stored frozen with protein inhibitors. Nuclear protein extraction. Frozen pellets were thawed and resuspended in 0.5 ml high-salt buffer (0.5 M NaCl, 0.05 M MgCl 2, 0.01 M Tris–HCl, pH 7.5, 10 mL/mL PMSF, 25 mL/mL DNase, 10 mL/mL of aprotinin, and 20 mL/mL of leupeptin) in a proportion of 2–3 3 10 7 nuclei/ml. Tubes were incubated at 37°C for 20 min, chilled on ice to stop reactions, and centrifuged at 12,000g (2300 rpm) for 10 min at 4°C. Supernatants were reserved as nuclear protein isolates. Immunodetection of PKC isoforms. Proteins (20 –100 mg) from cell fractions were electrophoretically separated on a 0.1% sodium dodecyl sulfate (SDS)–7.5% polyacrylamide slab minigel, and membrane proteins (50 mg) were fractionated with 6% SDS–PAGE, according to the method of Laemmli [21]. Proteins were transferred to a nitrocellulose membrane using an adapted technique from the original Western blot procedure [22]. Membranes were blocked with 5% Carnation low-fat milk in PBS containing 0.05% Tween-20 and incubated with rabbit polyclonal antibodies to isoform-specific peptides of the a, bI, bII, g, d, e, h, and z PKC (Oxford Biomedical Research Inc., Oxford, MI; R & D Antibodies, Berkeley, CA) or obtained from Alan Fields, University of Texas (Galveston) for 2 h at room temperature. Membranes were washed three times for 10 min with PBS–Tween solution, and then they were incubated 30 min with goat anti-rabbit (GAR) antibody conjugated to horseradish peroxidase (HDR) diluted 1:30,000 (Kirkegard and Perry, Gaithersburg, MD). After three brief washes with PBS–Tween solution, antigen– antibody complexes were detected using enhanced chemiluminescence (Amersham Life Science, Inc., Arlington Heights, IL) on autoradiographic film. Baculovirus expressed specific PKC isoforms (Oxford) were used as positive controls for the PKC antibodies. Antibodies have been characterized by their manufacturers to be PKC isoform specific. The molecular weights of bands were determined using protein molecular weight standards (Sigma Chemical Co., St. Louis, MO) that were electrophoretically separated and transferred at the same time as the protein samples. Protein blots were stained with 0.5% Ponceau S solution to confirm equal loading of the lanes. Transfection of GM86 cells with PKCa antisense. Human PKCa (obtained from American Type Culture Collection, Rockville, MD) was subcloned into the EcoRI site of pBluescript KS 1 (Stratagene, LaJolla, CA). (The homology between human and murine PKCa is 89%.) Digestion with HindIII was followed by further ligation and the selection of transformants with the PKCa cDNA in the antisense orientation. Lipofectin was used for transfections according to manufacturer’s instructions. Stable transfectants were selected in G418. PKCa protein expression was verified by Western blot analysis.
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cause PMA had no significant effect on PKCd and e, these isoforms were excluded from further consideration.
FIG. 1. Cytosolic and nuclear distribution of the a, bI, and bII isoforms of PKC. FELC were separated into their cytosolic and nuclear fractions, run on a 7.5% polyacrylamide gel, and then transferred to a nitrocellulose membrane. 5 3 106 cells were used for each of the samples. The samples were then probed with PKC isoformspecific antibodies for the indicated isoforms of PKC. N indicates the lanes containing the nuclear fractions, while C indicates the lanes containing the cytosolic fractions.
Untransfected or transfected (vector only or PKCa antisense) FELC were treated with 5 mM HMBA or 100 ng/ml PMA. Cultures were incubated for 96 h. Differentiation was evaluated by determining the percentage of benzidine positive cells [8]. RNA extraction and PCR determination of b-globin. For quantification of RNA using competitive PCR, 1 mg total RNA was reversetranscribed in a 20-ml reaction mixture using reverse transcriptase (Perkin-Elmer, Foster City, CA). The product was then subjected to competitive PCR to measure the b-globin transcription level using primers 59-GCTGAGAAGGCTGCTGTCT-39 (sense) and 59-ACCTTCTGGAAGGCAGCC-39 (antisense). As an internal control, RNA for the ubiquitous ribosomal protein S16 was also quantitated using 59-CTGGAGCCTGTTTTGTTCTG-39 (sense) and 59-TGAGATGGACTGTCGGATGG-39 (antisense). In a typical quantitation, a series of reactions was set up with increasing amounts of sample and concomitant decreasing amounts of standard (prepared as described below) at a twofold interval. The resulting products were analyzed on a 2% agarose gel and scanned using a laser densitometer (Molecular Dynamics, Los Alamos, CA). The condition at which the sample band and the standard band display similar intensities was used to determine the amount of corresponding cDNA in the PCR reaction [23]. Standard for competitive RT-PCR of the mouse b-globin was prepared from plasmid containing the mouse b-globin cDNA by PCR with primers 59-GCTGAGAAGGCTGCTGTCTACCCTTGGACCCAGCGGTACTTTGAT-39 (sense) and 59-ACCTTCTGGAAGGCAGCC-39 (antisense). The product of such a reaction serves as template for the original pair of b-globin primers yielding a shorter product (311 bp) distinguishable from the natural product (380 bp). Standard for the ribosomal protein S16 was generated similarly. The PCR products were gel purified, electroeluted, and the concentration determined by UV absorbance at 260 nm. The product was then coamplified in the quantitative RT-PCR reactions, as described above.
HMBA and PMA Induced Changes in PKC Isoform Expression Results shown in Table 1 confirm previous findings that PMA inhibits HMBA-induced differentiation in Friend erythroleukemia cells [7]. The localization of PKCa and bI to the nuclear, membrane, or cytosolic compartment was examined in response to HMBA or PMA treatment alone or in combination at 0, 0.5, 24, or 96 h. Figure 2A shows that nuclear localization of PKCa was maximal after 24 h of HMBA treatment, whereas nuclear PKC bI decreased throughout the treatment period (Fig. 2B). PKC bII was not further studied because of its weaker nuclear signal on immunoblot. With PMA treatment, the pattern of protein localization changed. Nuclear PKCa protein levels peaked at 0.5 h during PMA or combination treatment and were no longer observed at 24 or 96 h. Nuclear PKCbI protein levels decreased at 0.5 h and returned to control levels by 24 h. With combination treatment, PKCbI protein levels decreased at 0.5 h and were absent at 24 h, followed by a reappearance of protein expression at 96 h. Both cytosolic and membrane fractions of FELC subjected to the same treatments over time exhibited a downregulation of PKCa by 24 h that was maintained at 96 h (Figs. 3A and 3B). PKCa protein levels were elevated at the same time points in HMBA-treated cells. The combination of PMA and HMBA treatment resulted in total downregulation of PKCa at 96 h in the cytosolic, membrane, and nuclear compartments, suggesting that the ability of PMA to block HMBA-induced differentiation might be the result of a global downregulation in PKCa. HMBA-Induced Differentiation and Its Inhibition by PMA and PKCa Antisense Because PKCa protein localization to the nucleus occurred within the 24-h period of commitment, and
RESULTS
Cellular Distribution of PKC Isoforms in FELC The cytosol and nuclear fractions of Friend erythroleukemia cells in the logarithmic phase of growth were isolated and probed for PKC isoform expression. Although the a isoform was the most abundant, it was localized mainly in the cytosol fraction of the cells. The bI and bII isoforms, on the other hand, were found in the nucleus (Fig. 1), as well as cytosol. The novel PKC isoforms d and e were also examined. PKCe was only faintly detected in the cytosol and did not localize to the nucleus, and PKCd was absent from the cytosol and faintly detected in the nucleus (data not shown). Be-
TABLE 1 Differentiation in FELC Treated with HMBA Alone, PMA Alone, or a Combination of the Two Treatment
% Differentiation
Control HMBA PMA HMBA 1 PMA
0 6 2a 70 6 5 162 562
Note. Cells were treated for 96 h with HMBA (5 mM), PMA (100 ng/ml), or both. The percentage of benzidine cells was determined for four experiments. a Mean 6 SEM.
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FIG. 2. Expression of PKCa or PKCbI in the nucleus after treatment with 5 mM HMBA, 100 ng/ml PMA, or 5 mM HMBA 1 100 ng/ml PMA. FELC were treated with the chemicals for 0, 0.5, 24, or 96 h, and the nuclear fractions were then probed for the a isoform (A) or the bI isoform (B). Immunoblots are representative of three separate experiments.
because PMA downregulated PKCa protein levels by 24 h, we tested the effect of transfecting FELC with antisense PKCa or with vector alone. As shown in Fig. 4, transfection of FELC with PKCa antisense also inhibited HMBA-induced differentiation by 41%, as did PMA compared to untransfected cells or vector control cells (not shown). Inhibition of b-Globin Gene Expression with PKCa Antisense Transfection Because b-globin is the principal gene expressed during erythroid differentiation, we tested the hypothesis that PKCa-mediated inhibition of differentiation might affect the level of b-globin expression. As shown in Fig. 5, an 85% reduction of b-globin mRNA expression in PKCa antisense expressing cells was measured after 72 h of HMBA treatment, compared to cells transfected with vector alone. A 40% reduction, compared to vector control of b-globin mRNA, was seen even after 96 h of treatment. DISCUSSION
Depending on the cell type studied, phorbol esters can either induce differentiation or block terminal differentiation. Specific isoforms of PKC have been impli-
cated in these processes. The clearest association of PKC isoforms with proliferation and differentiation has been documented for human leukemia cell lines [2, 5, 6, 24, 25]. Calcium-dependent PKCa and PKCb have been associated with differentiation and proliferation, respectively, in human K562 erythroleukemia cells [6, 25]. Only PKCbII translocated to the nucleus in response to proliferative signals in K562 cells. Antisense PKCbII blocked proliferation. PMA-induced cytostasis was reversed upon removal of PMA, and resumption of proliferation was accompanied by reexpression of PKCbII to near control levels, whereas PKCa and z levels remained elevated for several days after removal of PMA. PMA treatment of human erythroleukemia cells causes megakaryocytic differentiation and cessation of proliferation, whereas sodium butyrate induces megakaryocytic differentiation without cytostasis and causes increases in both PKCa and PKCbII in whole cell lysates. We have found that a murine erythroleukemia cell line contains the a, bI, bII, d, and e isoforms of PKC. Each of these PKC isoforms has been implicated to some extent in proliferation/differentiation events of FELC. Most recently, we have shown that PKCb binds to DNA in FELC and is closely associated with proliferative events in these cells [26]. Several additional
FIG. 3. Effect of HMBA and PMA on cytosolic and membrane localization of PKCa. FELC were treated with 100 ng/ml PMA, 5 mM HMBA, or both for the time indicated. (A) Immunoblot of cytosolic fraction or (B) membrane fraction. Representative of three experiments.
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FIG. 4. HMBA-induced differentiation of FELC. Wild type FELC were either treated with 5 mM HMBA (bar labeled Untransfected) to induce differentiation or 5 mM HMBA 1 100 ng/ml PMA (bar labeled PMA) to inhibit the HMBA effect. HMBA (5 mM) was added to “vector only” transfected cells or cells that were transfected with the antisense cDNA to PKCa (bar labeled Antisense). Differentiated cells were scored after 96 h of incubation by benzidine staining. Mean (6 SE) values: 78.9 6 1.3% for wild type cells treated with HMBA only (versus 72 6 1.5% for vector transfected cells), 1.5 6 0.2% for cells that were treated additionally with PMA, and 33 6 2.1% for PKCa antisense transfected cells (n 5 3).
studies have demonstrated that PKC activation is important early in HMBA-induced differentiation followed by PKC downregulation [7, 11, 13–14]. Leng et al. [14] found that translocation of the d and e PKC isoforms from the soluble portion of the cell to the particulate portion occurred after 15–30 min of stimulation with HMBA. Although we confirmed a translocation of the d and e PKC isoforms from the soluble to particulate fraction of FELC within 30 min of stimulation with HMBA, we found no nuclear localization of these isoforms in response to HMBA treatment. In contrast, the localization of the predominant PKCa isoform to the nucleus in a time-dependent manner coincident with the commitment phase of differentiation was pursued in terms of the effect of phorbol ester to block this HMBA-induced differentiation event. Changes in nuclear PKCbI were observed but because constitutive expression was seen in untreated cells, changes were not pursued in terms of correlations with the differentiation program. Although nuclear PKC protein activity and expression have been correlated with cell differentiation in
other cell types [24, 27, 30], very few studies have addressed the temporal relationship between PKCa isoform localization to the nucleus and commitment to differentiation. The ability of phorbol ester to reverse HMBA-induced differentiation of FELC has never been adequately explained. Although changes in diacylglycerol and PKC have been correlated with the inhibition of differentiation, the role of specific PKC isoform localized to the nucleus had not been previously addressed. Because cell lines and their variants differ in their PKC isoform distribution and their ability to differentiate in response to phorbol esters, it will be necessary to study a number of model systems before a clear picture of the molecular details of signaling pathways critical for these events can be ascertained. In the GM-86 clone of FELC, which is a growth factor independent, constitutively proliferating cell line, the cytosol and nuclear distribution of various PKC isoforms indicates that the more abundant PKCa isoform is primarily located in the cytosol, suggesting inactivation, while the bI and bII isoforms are primarily in the nucleus. The nature of their temporal distribution in response to the induction of differentiation and its inhibition provides support for the hypothesis that PKCa is critical for erythroid cell differentiation and PKCb isoforms are important for erythroid cell proliferation [6, 25, 26, 28]. The isoforms critical for regulating these biological events are likely to localize to the appropriate compartments in the cell where essential substrates are located. Our focus on the nuclear compartmentalization of PKC isoforms has been influenced by our findings, as well as others, that PKCb isoforms are
FIG. 5. b-Globin RNA expression during HMBA-induced differentiation. HMBA (5 mM) was used to induce differentiation in wild type (WT, short dashed line), vector transfected (V, long dashed line), or PKCa antisense transfected (A, solid line) cells. b-Globin mRNA expression was measured at 24-, 48-, 72-, and 96-h time points. b-Globin mRNA expression was measured using RT-PCR and is expressed as a ratio, compared to S16 mRNA.
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localized to the nucleus in response to erythroid cellspecific growth factors, such as erythropoietin, IL-3, and GM-CSF [31–32]. Although activity levels of PKC were not assessed in the current study, earlier studies from our laboratory have reported nuclear PKC activity in FELC treated with HMBA or PMA [28]. The greatest activity in the nucleus was seen in logarithmically growing cells, particularly those treated with PMA. The finding that PKCa localization to the nucleus is an early event in erythroid cell differentiation and is critical for b-globin expression supports the hypothesis that PKCa is an important component of the signal transduction pathway to gene expression. The ability of PKCa antisense transfection of FELC to block HMBA-induced differentiation, as did PMA, further supports an important role for PKCa in differentiation. REFERENCES 1.
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