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AP-1 Activity Affects the Levels of Induced Erythroid and Megakaryocytic Differentiation of K562 Cells
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Vol. 352, No. 2, April 15, pp. 298–305, 1998 Article No. BB980597
AP-1 Activity Affects the Levels of Induced Erythroid and Megakaryocytic Differentiation of K562 Cells1 Dan Rosson2 and Thomas G. O’Brien The Lankenau Medical Research Center, 100 Lancaster Avenue, Wynnewood, Pennsylvania 19096-3411
Received October 9, 1997, and in revised form January 12, 1998
The effect of modulating AP-1 activity on the levels of induced erythroid and megakaryocytic differentiation of the erythroleukemia cell line K562 was examined. Cells were stably transfected with expression vectors encoding either a tetracycline-responsive dominant negative c-Jun (JunDN) or a hybrid Raf protein inducible by estrogen. Down-regulation of AP-1 activity by induction of JunDN enhanced erythroid differentiation by two agents, cytosine arabinoside and activin A. Induction of AP-1 activity by elevated Raf activity inhibited erythroid differention, thus mimicking the well-known effect of tetradecanoyl phorbol acetate (TPA) on this process. Induced Raf activity also brought about partial megakaryocytic differentiation of the line. However, inhibition of TPA-induced AP-1 activity by induction of JunDN gave mixed results. While the cytological effects of TPA treatment observed on cytochemical staining were inhibited by JunDN , two protein markers for megakaryocytic differentiation were increased. These results, while supportive of current models of hematopoietic lineagespecific gene expression, suggest a complex and temporal mechanism of lineage commitment. q 1998 Academic Press
Key Words: AP-1; K562; hematopoietic cell differentiation.
The K562 cell line is a useful model system for studies of cellular differentiation because of its potential to differentiate along two cell lineages (1). Erythroid differentiation of the cell line can be induced by a number of compounds, including cytosine arabinoside (ara
1 This work was supported by National Institutes of Health Grants CA36353 and CA72609 from the National Cancer Institute. 2 To whom correspondence should be addressed. Fax: (610) 6452205.
C)3 (2) and activin A (3, 4). On the other hand, upon TPA treatment, the cells assume an adherent, spindleshaped morphology, while expressing proteins associated with the megakaryocytic lineage (1, 5). Thus, while the cell line is malignant and may have properties different from normal bone marrow stem cells, it nevertheless offers the opportunity to study both the signal transduction events involved in differentiation and the molecular events involved in lineage commitment. The processes of differentiation and lineage commitment are controlled by transcription factors, which, in turn, control which lineage-specific genes are expressed (6, 7). Recently, the relative levels and the activity of several families of key transcription factors have been implicated to be at least partly at play in the process of controlling the lineage along which hematopoietic cells differentiate. The members of these families interact among each other via leucine zipper motifs to generate the active dimeric species. One specific model proposes that the transcription factors AP-1 and NF-E2 compete for the same binding site in the promoters of certain lineage-specific genes (8–10). This was based, in part, on the finding that TPA treatment of K562 cells results in elevated AP-1 binding to an erythroidspecific gene promoter and in a decreased level of NFE2 binding. The relative activity level of each factor determines the lineage along which the cell will differentiate and the activity levels are, in turn, controlled by the relative levels and states of phosphorylation of each component of the factors. In this model, a predominance of NF-E2 over AP-1 promotes erythroid differentiation, whereas AP-1 suppresses it. An extension of this model would be that AP-1 activity not only suppresses erythroid-specific genes but induces megakary3 Abbreviations used: ara C, cytosine arabinoside; TPA, tetradecanoyl phorbol acetate; MAPK, mitogen-activated protein kinase; JunDN , dominant negative Jun; tet, tetracycline; ER, estrogen receptor; TRE, TPA-response element; PKC, protein kinase C.
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ocytic-specific genes. This model predicts that stimulation of Raf activity should mimic TPA treatment by stimulating AP-1 activity via the MAPK pathway and hence inducing megakaryocytic differentiation. Furthermore, the model also predicts that inhibition of AP1 activity would suppress TPA-induced megakaryocytic differentiation, while augmenting, if not inducing, erythroid differentiation. We have examined these issues by constructing sublines of K562 with inducible genes encoding either a constitutively active Raf or a dominant negative Jun (JunDN). Our results show that, as expected, stimulation of Raf activity did indeed stimulate megakaryocytic differentiation, while suppressing induced erythroid differentiation. Furthermore, JunDN enhanced erythroid differentiation. However, unexpectedly, JunDN expression inhibited some aspects of TPA-induced megakaryocytic differentiation while enhancing others. These results are discussed in the context of an overall model for the regulation of tissue-specific gene expression and differentiation of erythroid cells by relevant transcription factors. MATERIALS AND METHODS Cell culture and DNA transfections. K562 cells were purchased from the American Type Culture Collection and cultured in RPMI medium supplemented with 10% fetal bovine serum plus antibiotics. In order to express junDN in an inducible manner, the tet-responsive system of Gossen and Bujard (12) was used. Stable transfectants expressing the tet-responsive transactivating protein tTA were obtained by electroporating 10 mg of the tTA expression plasmid pUHD13-3 along with a plasmid conferring hygromycin resistance in a suspension of phosphate-buffered saline using a Bio-Rad gene pulser equipped with a capacitance extender. One pulse of 300 V at 250 mF was used. After 10 min, the cells were suspended in culture medium, incubated for 1 day, adjusted to 200 mg hygromycin/ml, and transferred to multiwell plates. After approximately 4 weeks of incubation drug-resistant clones were transferred out of the multiwell plates, expanded in the absence of hygromycin, and analyzed for tet-responsive expression of tTA. A suitable subclone, K562tTA, was chosen for future constructions. The reading frame of junDN was constructed by PCR deletion mutagenesis using full-length c-jun as described (12). The resulting PCR fragment containing a 5* deletion and flanked by EcoRI sites was cloned into the EcoRI site of pUHD10-3. The resulting plasmid, pUHD10-3junDN , was electroporated as described above into K562tTA using pSVzeo as a resistance marker and 350 mg Zeocin (Invitrogen)/ml for selection. After isolation and expansion, clones were analyzed for junDN expression by Western analysis. pLNCXDraf-1:ER (13) was generously supplied by Dr. Martin McMahon of DNAX Research Institute of Molecular and Cellular Biology. Infectious retrovirus was generated by first transfecting the ecotropic packaging cell line GP/E86 and then using virus-containing supernatant to infect the amphotropic cell line PA317. Supernatant from this infection was used as high-titer virus stock to infect K562 cells while using 1 mg of G418/ml for selection. Clones expressing Draf-1:ER were identified by Western analysis using anti-estrogen receptor (ER) antibodies (Santa Cruz). Western analysis. Cell pellets were lysed in Tris–Cl buffer containing 0.1% SDS and proteinase inhibitors. DNA was sheared to reduce viscosity, and protein concentration was determined with Bio-
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Rad protein assay dye reagent. Fifty micrograms of total protein was mixed with an equal volume of 21 sample buffer [0.125 M Tris– Cl (pH 6.8), 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.002% bromophenol blue]. Samples were heated in a boiling water bath for 5 min and then loaded onto a denaturing 6% polyacrylamide electrophoresis gel. After separation, proteins were electrophoretically transferred to a nitrocellulose membrane. The filters were first treated with Trisbuffered saline [50 mM Tris–Cl (pH 7.5), 0.15 M NaCl] with 0.05% Tween 20 (TBST) containing 3% nonfat milk. This was followed by incubation in 5 ml of TBST with 1% bovine serum albumin per milliliter and anti-Jun antibodies (No. SC44; Santa Cruz), anti-ER (No. SC543; Santa Cruz), and/or anti-a2 integrin antibody (Chemicon) at concentrations recommended by the manufacturers for 5 h at room temperature. The blots were subsequently washed in TBST and then incubated with the appropriate immunoglobulin peroxidase-conjugated antibodies (Amersham) for 1 h. After being washed in TBST, the blots were developed using the enhanced chemiluminescence Western immunoblotting system (Amersham). Analysis of the platelet fibrinogen receptor complex IIb–IIIa was performed similarly, except that 15 mg of total protein was used for electrophoresis and 2-mercaptoethanol was omitted from the sample buffer. Antibodies to Ilb–Illa were polyclonal rabbit serum (14) provided by Dr. Karen Knudsen of Lankenau Medical Research Center. Analysis of cellular differentiation. Erythroid differentiation was induced by adjusting logarithmically growing cells to either 0.36 mM ara C or 60 ng/ml activin A. After 5 days, the cells were stained with a 1/4 volume of benzidine solution (0.04% 3,3 *,5,5*-tetramethylbenzidine, 2% hydrogen peroxide, 12% acetic acid). Positive (blue-staining) cells were counted under a hemacytometer. A total of at least 200 cells were counted. Megakaryocytic differentiation was induced by adjusting cell cultures to 1008 M TPA for 2 days. Differentiation was assessed by cellular morphology, Wright–Giemsa staining, and Western analysis of the differentiation antigens Ilb–Illa and the a2 integrin. When estrogen was present in cultures, its concentration was 1 mM. AP-1 analysis. Measurement of AP-1 activity levels was assessed by transiently transfecting cells with pTRE-luc (15) (provided by Michael Karin of the University of California, San Diego) by the electroporation procedure described above. AP-1 activity induces luciferase activity from this construct by means of an artificial TPAresponse element (TRE) located in its promoter. After transfection, the cells were divided into two equal aliquots and one was adjusted to either 1008 M TPA or 1 mM estrogen. Subsequently, both were added to 10 ml of medium and incubated for 24 h. Cells were lysed in buffer containing 1% Triton X-100, and luciferase activity was measured on a Monolight 2010 luminometer (Analytical Luminescence Laboratory). All results presented are representative of at least three experiments.
RESULTS
Because randomly isolated sublines can vary from the parental line in the rate or extent of differentiation, there is a consequent difficulty in attributing the expression of an exogenous gene to a particular phenotypic change. These difficulties must be overcome by examining a large enough number of clones, with and without the exogenous gene, to collect statistically significant data. In cases in which partial effects are seen and experimental variation is problematic, the number of clones necessary to achieve such data can be large. We chose to avoid these difficulties by the use of two effective inducible systems in which the activity of the
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FIG. 1. Western Analysis of K562 transfectants expressing exogenous JunDN and DRaf-1:ER. Whole-cell protein extracts were prepared, subjected to electrophoresis and blotting, and analyzed with anti-Jun (A) or anti-ER (B) antibodies as described under Materials and Methods. Sources of extracts are as follows: (A) Lane 1, K562; lane 2, K562junDN (0tet); lane 3, K562junDN (/tet). (B) Lane 1, K562; lane 2, K562Draf-1:ER (0estrogen); lane 3, K562Draf-1:ER (/estrogen).
exogenous gene could be modulated within the same line. In one system the activity of c-Jun was modulated by the expression of a truncated c-jun gene, junDN , which encodes a product possessing the protein’s DNA binding and protein dimerization domains but lacking the transactivating domain. This construct has been shown to possess dominant-negative activity toward c-Jun or AP-1 (12). Inducible junDN expression was obtained by means of the tetracycline-responsive expression vector of Gossen and Bujard (12). In order to establish a subline of K562 expressing an inducible raf gene, we infected K562 cells with a retrovirus expressing the Draf1:ER gene of Samuels et al. (13). The active protein transduced by this construct is a hybrid protein containing sequences encoding a constitutively active Raf fused to the hormone binding domain of the estrogen receptor. The Raf sequences are a truncated c-Raf-1 similar to v-Raf and hence require no activation from upstream signal transduction. Raf activity in this construct is inhibited by the downstream ER sequences. However, in the presence of estrogen, the hormone binding sequences of ER assume a different conformation and cease to inhibit Raf activity. Figure 1A shows Western analysis of K562 and one of the K562junDN transfectants which were obtained. All transfectants expressed a 30-kDa dominant-negative protein (lane 2) which responded to tetracycline by decreased synthesis of JunDN to almost undetectable levels (lane 3). The antibody raised to C-terminal amino acids necessary to detect the N-terminal truncated protein did not detect endogenous c-Jun. Figure 1B shows Western analysis of K562Draf-1:ER with and without estrogen. The antibody raised to the hormone binding domain of the estrogen receptor detects a major protein band of approximately 67 kDa as originally re-
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ported by Samuels et al. (13). Also consistent with the original study, activation of the protein’s enzymatic activity by estrogen resulted in an increased transcriptional activation of the gene. Figure 2 shows the effect of the two exogenous genes on AP-1 activity. Cells were transiently transfected with the plasmid TRE-luc (15), which contains a luciferase reporter gene under the control of the collagenase promoter containing a single artificial TRE. Parental cells treated with TPA showed a 250-fold increase in AP-1 activity over untreated cells. There are two salient features of the figure. First, TPA treatment could be mimicked in K562Draf-1:ER by treatment with estrogen. Second, in K562junDN cells, junDN expression reduced AP-1 activity from 250-fold to 16-fold over cells not treated with TPA. Addition of tetracycline to the media restored a significant amount of AP-1 activity, eliminating the possibility that clonal variation incurred in the construction of K562junDN cell line could account for the response. We next used the K562junDN and K562Draf-1:ER clones to examine the effect of modulating AP-1 activity on induced differentiation. All K562junDN clones have a normal morphology and a growth rate which is unaltered by the level of junDN expression. However, the response to erythroid differentiation induced by either ara C or activin A was significantly affected by the presence of tetracycline. As seen in Fig. 3, ara C induced hemoglobin synthesis in the parental clone to the extent that approximately 65% of the cells stained blue with benzidine reagent. A lesser extent of hemoglobinization was seen in K562junDN cells in the presence of tet. This could be due either to a small amount
FIG. 2. AP-1 activity levels. Cell lines were transiently transfected with TRE-luc and analyzed for AP-1-induced luciferase activity as described under Materials and Methods. The relative luciferase activity is shown for the three cell lines under the indicated conditions.
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FIG. 3. Levels of erythroid differentiation induced by ara C or activin A as assessed by benzidine staining. Cells grown under the indicated conditions were stained with benzidine to assess hemoglobin levels as described under Materials and Methods. The percentage of benzidinepositive (blue-staining cells) under each condition is shown.
of JunDN expression in the presence of tet or to clonal variation between K562junDN and the parental line. However, the percentage of benzidine-positive cells was increased markedly in the K562junDN cells grown in the absence of tet. Comparable results were obtained when activin A was used as the inducer. Treatment of parental cells with this cytokine resulted in 30% of the cells differentiating. Again, the K562junDN clone exhibited a significantly higher level of differentiation with junDN induced over cells with junDN suppressed. Also shown in Fig. 3, treatment of K562Draf-1:ER cells with ara C resulted in 70% of the cells becoming benzidine positive, whereas the stimulation of Draf-1:ER activity by addition of 1 mM estrogen mimicked TPA treatment of K562 and thus almost completely inhibited this differentiation. Similar results were obtained when activin A was used as the inducer. All of these results are consistent with the concept that AP-1 activity inhibits erythroid differentiation. We next examined the effects of modulating AP-1 activity on TPA-induced megakaryocytic differentiation. Among the morphological changes induced by TPA treatment of K562 is a larger average cell volume and a spindle-shaped morphology among some of the cells in culture. The percentage of adherent cells also increases. Expression of junDN virtually eliminated the appearance of spindle-shaped cells induced by TPA treatment. When tet was added to the media of K562junDN , some of the spindle-shaped morphology inducible by TPA treatment was restored. However, the levels were not that of wild-type K562. As seen in Fig. 4, when analyzed cytochemically by Wright–Giemsa staining, untreated K562 cells show a relatively homogeneous population of blastlike cells with many cytoplasmic protrusions, a high (ú1) nuclear-to-cytoplasmic ratio, and round nuclei. Upon TPA-induced differentiation, the cell population progresses to one of more mature cells with a megakaryocyte morphology characterized by a lower (°1) nuclear-to-cytoplasmic ratio, a lobulated nucleus resulting from endomitosis,
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and a highly vacuolated cytoplasm. Also present are cells with a histiocytoid morphology. These are cells which appear spindle-shaped under light microscopy. They are characterized by a large acidophilic nucleus and lightly staining cytoplasm. No mitotic cells are evident. As expected, induction of AP-1 activity via induction of Raf activity in K562Draf-1:ER resulted in the cells assuming a more differentiated morphology. A quantitation of this change is presented in Table I. The extent of differentiation was not as great as that elicited by TPA treatment. Cell size increased similarly but cytoplasmic vacuolization was less extensive and some mitotic cells were still present. Consistent with the lack of spindle-shaped cells viewed under light microscopy, no histiocytic cells were apparent on Wright– Giemsa stains. As expected, expression of junDN decreased the above morphological changes induced by treatment with TPA. Cell size was still markedly increased and vacuolization was extensive. However, few histiocytic cells were evident. This is in contrast to K562junDN cells with junDN down-regulated by the presence of tet. Cytochemical analysis of cells in this case showed more abundant histiocytoid cells. In order to carry the analysis of TPA-induced differentiation further, the levels of megakaryocytic-associated proteins were measured. The platelet fibrinogen receptor complex antigen II–IIIa is a glycoprotein complex associated with platelets and megakaryocytes and is an early marker for differentiation along the megakaryocytic lineage (5). Fig. 5A shows Western blot analysis of IIb–IIIa in K562 and its derivatives using anti IIb–IIIa antibody (14). TPA treatment of K562 induced the synthesis of IIb–IIIa (lane 2), and as expected, induction of Raf activity in K562Draf-1:ER also induced the appearance of the IIb–IIIa antigen (lane 7). However, in contrast to the results obtained on cytochemical analysis, the inhibition of AP-1 by JunDN increased rather than decreased the level of IIb–IIIa expression (lanes 4 and 5). Similarly, the levels of the a2 integrin, which serves as a surface receptor for collagen on plate-
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FIG. 4. Wright – Giemsa staining of K562 cells and derivatives. The panel labeled K562 indicates untreated K562 cells. Immediately below are K562 cells treated with TPA. The middle two panels are K562junDN cells treated with TPA in the absence and the presence of tet. The right panels indicate K562Draf-1:ER cells without and with estrogen treatment.
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AP-1 ACTIVITY AND DIFFERENTIATION OF K562 CELLS TABLE I
Cytological Analysis of K562, K562junDN , and K567Draf-1:ER Before and After Treatment with TPA or Estrogena
a
Cell line
% Megakaryoblast
% Megakaryocyte
% Histiocytic
% Mitotic
K562 K562 / TPA K562junDN K562junDN/TPA K562junDN/tet/TPA K562Draf-1:ER0estrogen K562Draf-1:ER/estrogen
90 5 92 5 1 95 20
7 60 5 91 49 1 72
1 35 1 4 50 2 7
2 2
2 1
Cytochemical analysis was assessed by classification of cells into one of four categories based on criteria described in the text.
lets and other cells, were also examined. This megakaryocytic marker was also induced by Raf activity as in TPA treatment of K562 cells. Consistent with the experiments measuring IIb–IIIa levels, a2 integrin levels were also increased with junDN expression compared to the same cells with junDN down-regulated by tet (Fig. 5B). These results are in contrast to the results obtained by morphological and cytochemical analysis and indicate that while decreased AP-1 activity is associated with erythroid differentiation, induction of megakaryocytic differentiation is more complex than simply an increase in AP-1 activity. DISCUSSION
Experimental evidence suggesting that modulation of AP-1 activity is involved in erythroid differentiation dates back to work by Rovera et al. (16), who showed that erythroid differentiation of spontaneously differentiating sublines of F-MEL cells was abolished by TPA. This work was extended by Yamasaki et al. (17), who showed that TPA also inhibited hexamethylenebi-
FIG. 5. Western analysis of K562 cells and derivatives probed for IIb–IIIa expression (A) and a integin expression (B). Sources of extracts are as follows: (A) lane 1, untreated K562 cells; lane 2, K562 cells treated with TPA; lane 3, untreated K562junDN ; lane 4, K562junDN cells treated with TPA in the absence of tet; lane 5, K562junDN cells treated with TPA in the presence of tet; lane 6, K562Draf-1:ER cells in the absence of estrogen; lane 7, K562Draf1:ER cells in the presence of estrogen. (B) Extracts are the same as in (A).
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sacetamide-induced differentiation of F-MEL cells. The molecular basis for these observations lies in the mode of action of TPA. TPA is now known to be a potent inducer of AP-1 activity via activation of PKC (18). AP1 binds to the promoter sequence TGACTCA, also known as the TPA-response element (19), and thereby increases the expression of target genes responsible for most of the phenotypic changes elicited by TPA treatment. While increases in AP-1 activity are associated with decreased erythroid differentiation, there is also experimental evidence for the converse. Retinoic acid, an inducer of erythroid differentiation in one cell line (20), is an antagonist of AP-1 activity (21, 22). The mechanism of this inhibition probably involves a competition of these proteins for common coactivators (23). Additional evidence is provided by Francastel et al. (24), who studied a variant of F-MEL cells which was resistant to induced differentiation. They found that the resistance was associated with increased levels of cJun protein and that expression of anti-sense c-jun RNA could alleviate this inhibition. We have extended this association by experimentally modulating AP-1 activity by introduction of an inactive competitor to one of the AP-1 components, c-Jun. The truncated, dominant negative protein competes with the endogenous functional c-Jun for binding sites in c-Fos, thereby decreasing the concentrations of functional Fos/Jun dimers. This was seen in the initial experiments in which luciferase activity controlled by an AP-1-responsive promoter was significantly reduced by JunDN levels. Consistent with those previous studies cited above, downregulation of AP-1, while failing to initiate erythroid differentiation by itself, nevertheless, increased the extent of erythroid differentiation induced by two agents, ara C and activin A. To avoid any ambiguity in interpretation of the data, we eliminated the possibility of clonal variation by use of an effective inducible system. We found that the extent of induced erythroid differentiation could be reproducibly reversed within the same
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subline by adding or subtracting tet to the media of the cells. The involvement of AP-1 activity in the process of differentiation was further supported by experiments with a subclone of K562 expressing an inducible version of c-Raf. Cells with induced Raf activity showed elevated levels of AP-1, as assessed by luciferase activity under the control of a TRE element. This AP-1 activity, as in the case of TPA-induced AP-1 activity, not only inhibited erythroid differentiation induced by the two agents, but also elicited megakaryocytic differentiation. While this work was in progress, Whalen et al. (25) published similar results using constitutively active MAPK kinase expression vectors to induce megakaryocytic differentiation. The data discussed so far are consistent with the simple concept that decreases in AP-1 activity favor erythroid differentiation, whereas elevated levels favor megakaryocytic differentiation. However, this concept was not supported in experiments in which TPA treatment was used to induce megakaryocytic differentiation of K562 derivatives expressing JunDN . While JunDN expression inhibited the morphological changes induced by TPA treatment as expected, the gene product actually increased expression of two protein markers associated with the megakaryocytic phenotype. This observation indicates that control of lineage commitment is not as simple as modulating either the absolute level of AP-1 or the relative level of AP-1/NF-E2. This finding was perhaps presaged in previous work showing that NF-E2 is not exclusively an erythroid transcription factor (26, 27). While binding to NF-E2 sites in DNA appears to undergo a shift from NF-E2 to AP-1 binding upon TPA treatment, NF-E2 p45 mRNA is nevertheless increased (10). Similarly, knockout mice for the NF-E2 gene die of thrombocytopenia, while erythropoiesis is relatively normal (28). The control of lineage commitment thus appears to be a more complex and perhaps temporal mechanism. Differentiation of the cell culture along either lineage takes place over a period of several days. However, the expression and the activity of some key signal transduction components vary during the course of differentiation. For example, dimethyl sulfoxide or HMBA treatment of F-MEL cells induces an immediate increase in membrane-bound PKC activity (29–31). Depending on the cell line, this increased activity occurs by translocation of cytosolic PKC to the membrane or the activation of inactive membrane-bound PKC (32). In either case, this rise in activity is followed by a sustained decrease in total PKC activity occurring over the 5-day period of differentiation. Another time-dependent variable in differentiation events involves relative levels of the various AP-1 components. While the predominate members of AP-1 are often considered to be c-Fos and c-Jun, both are mem-
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bers of families of related proteins. The Fos family consists of c-Fos, Fos B, and Fra-1, while the Jun family consists of c-Jun, Jun-B, and Jun-D (33, 34). In some cell lines, Fos/Jun can induce the synthesis of Fra-1 (35). Serum growth factors stimulate transcription of fra-1 with delayed and protracted kinetics compared to c-fos and fos B induction (36, 37). Fra-1/Jun dimers bind with equal affinity to TREs but do not transactivate target gene expression as do Fos/Jun (38). Thus, Fos/Jun can, in some instances, induce the synthesis of their own inhibitor. To more fully characterize the molecular mechanisms of lineage commitment, the levels of NF-E2, Maf proteins, and AP-1 components will have to be followed throughout the course of differentiation. ACKNOWLEDGMENT The authors thank the people at Genetech for their generosity in supplying the activin A used in this study.
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20. Garg, L. C., and Brown, J. C. (1983) Differentiation 25, 79–83. 21. Schule, R., Rangarajan, P., Yang, N., Kliewer, S., Ransone, L. J., Bolado, J., Verma, I. M., and Evans, R. M. (1991) Proc. Natl. Acad. Sci. USA 88, 6092–6096. 22. Nicholson, R. C., Mader, S., Nagpal, S., Leid, M., Rochette-Egly, C., and Chambron, P. (1990) EMBO J. 9, 4443–4454. 23. Kamei, Y., Xu, L., Heinzel, T., Torchia, J., Kurokawa, R., Gloss, B., Lin, S.-C., Heyman, R. A., Rose, D. W., Glass, C. K., and Rosenfeld, M. G. (1996) Cell 85, 403–414. 24. Francastel, C., Groisman, R., Pfarr, C. M., and Robert-Lezenes, J. (1994) Oncogene 9, 1957–1964. 25. Whalen, A. M., Galasinski, S. C., Shapiro, P. S., Nahreini, T. S., and Ahn, N. G. (1997) Mol. Cell. Biol 17, 1947–1958.
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Vol. 352, No. 2, April 15, pp. 298–305, 1998 Article No. BB980597
AP-1 Activity Affects the Levels of Induced Erythroid and Megakaryocytic Differentiation of K562 Cells1 Dan Rosson2 and Thomas G. O’Brien The Lankenau Medical Research Center, 100 Lancaster Avenue, Wynnewood, Pennsylvania 19096-3411
Received October 9, 1997, and in revised form January 12, 1998
The effect of modulating AP-1 activity on the levels of induced erythroid and megakaryocytic differentiation of the erythroleukemia cell line K562 was examined. Cells were stably transfected with expression vectors encoding either a tetracycline-responsive dominant negative c-Jun (JunDN) or a hybrid Raf protein inducible by estrogen. Down-regulation of AP-1 activity by induction of JunDN enhanced erythroid differentiation by two agents, cytosine arabinoside and activin A. Induction of AP-1 activity by elevated Raf activity inhibited erythroid differention, thus mimicking the well-known effect of tetradecanoyl phorbol acetate (TPA) on this process. Induced Raf activity also brought about partial megakaryocytic differentiation of the line. However, inhibition of TPA-induced AP-1 activity by induction of JunDN gave mixed results. While the cytological effects of TPA treatment observed on cytochemical staining were inhibited by JunDN , two protein markers for megakaryocytic differentiation were increased. These results, while supportive of current models of hematopoietic lineagespecific gene expression, suggest a complex and temporal mechanism of lineage commitment. q 1998 Academic Press
Key Words: AP-1; K562; hematopoietic cell differentiation.
The K562 cell line is a useful model system for studies of cellular differentiation because of its potential to differentiate along two cell lineages (1). Erythroid differentiation of the cell line can be induced by a number of compounds, including cytosine arabinoside (ara
1 This work was supported by National Institutes of Health Grants CA36353 and CA72609 from the National Cancer Institute. 2 To whom correspondence should be addressed. Fax: (610) 6452205.
C)3 (2) and activin A (3, 4). On the other hand, upon TPA treatment, the cells assume an adherent, spindleshaped morphology, while expressing proteins associated with the megakaryocytic lineage (1, 5). Thus, while the cell line is malignant and may have properties different from normal bone marrow stem cells, it nevertheless offers the opportunity to study both the signal transduction events involved in differentiation and the molecular events involved in lineage commitment. The processes of differentiation and lineage commitment are controlled by transcription factors, which, in turn, control which lineage-specific genes are expressed (6, 7). Recently, the relative levels and the activity of several families of key transcription factors have been implicated to be at least partly at play in the process of controlling the lineage along which hematopoietic cells differentiate. The members of these families interact among each other via leucine zipper motifs to generate the active dimeric species. One specific model proposes that the transcription factors AP-1 and NF-E2 compete for the same binding site in the promoters of certain lineage-specific genes (8–10). This was based, in part, on the finding that TPA treatment of K562 cells results in elevated AP-1 binding to an erythroidspecific gene promoter and in a decreased level of NFE2 binding. The relative activity level of each factor determines the lineage along which the cell will differentiate and the activity levels are, in turn, controlled by the relative levels and states of phosphorylation of each component of the factors. In this model, a predominance of NF-E2 over AP-1 promotes erythroid differentiation, whereas AP-1 suppresses it. An extension of this model would be that AP-1 activity not only suppresses erythroid-specific genes but induces megakary3 Abbreviations used: ara C, cytosine arabinoside; TPA, tetradecanoyl phorbol acetate; MAPK, mitogen-activated protein kinase; JunDN , dominant negative Jun; tet, tetracycline; ER, estrogen receptor; TRE, TPA-response element; PKC, protein kinase C.
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ocytic-specific genes. This model predicts that stimulation of Raf activity should mimic TPA treatment by stimulating AP-1 activity via the MAPK pathway and hence inducing megakaryocytic differentiation. Furthermore, the model also predicts that inhibition of AP1 activity would suppress TPA-induced megakaryocytic differentiation, while augmenting, if not inducing, erythroid differentiation. We have examined these issues by constructing sublines of K562 with inducible genes encoding either a constitutively active Raf or a dominant negative Jun (JunDN). Our results show that, as expected, stimulation of Raf activity did indeed stimulate megakaryocytic differentiation, while suppressing induced erythroid differentiation. Furthermore, JunDN enhanced erythroid differentiation. However, unexpectedly, JunDN expression inhibited some aspects of TPA-induced megakaryocytic differentiation while enhancing others. These results are discussed in the context of an overall model for the regulation of tissue-specific gene expression and differentiation of erythroid cells by relevant transcription factors. MATERIALS AND METHODS Cell culture and DNA transfections. K562 cells were purchased from the American Type Culture Collection and cultured in RPMI medium supplemented with 10% fetal bovine serum plus antibiotics. In order to express junDN in an inducible manner, the tet-responsive system of Gossen and Bujard (12) was used. Stable transfectants expressing the tet-responsive transactivating protein tTA were obtained by electroporating 10 mg of the tTA expression plasmid pUHD13-3 along with a plasmid conferring hygromycin resistance in a suspension of phosphate-buffered saline using a Bio-Rad gene pulser equipped with a capacitance extender. One pulse of 300 V at 250 mF was used. After 10 min, the cells were suspended in culture medium, incubated for 1 day, adjusted to 200 mg hygromycin/ml, and transferred to multiwell plates. After approximately 4 weeks of incubation drug-resistant clones were transferred out of the multiwell plates, expanded in the absence of hygromycin, and analyzed for tet-responsive expression of tTA. A suitable subclone, K562tTA, was chosen for future constructions. The reading frame of junDN was constructed by PCR deletion mutagenesis using full-length c-jun as described (12). The resulting PCR fragment containing a 5* deletion and flanked by EcoRI sites was cloned into the EcoRI site of pUHD10-3. The resulting plasmid, pUHD10-3junDN , was electroporated as described above into K562tTA using pSVzeo as a resistance marker and 350 mg Zeocin (Invitrogen)/ml for selection. After isolation and expansion, clones were analyzed for junDN expression by Western analysis. pLNCXDraf-1:ER (13) was generously supplied by Dr. Martin McMahon of DNAX Research Institute of Molecular and Cellular Biology. Infectious retrovirus was generated by first transfecting the ecotropic packaging cell line GP/E86 and then using virus-containing supernatant to infect the amphotropic cell line PA317. Supernatant from this infection was used as high-titer virus stock to infect K562 cells while using 1 mg of G418/ml for selection. Clones expressing Draf-1:ER were identified by Western analysis using anti-estrogen receptor (ER) antibodies (Santa Cruz). Western analysis. Cell pellets were lysed in Tris–Cl buffer containing 0.1% SDS and proteinase inhibitors. DNA was sheared to reduce viscosity, and protein concentration was determined with Bio-
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Rad protein assay dye reagent. Fifty micrograms of total protein was mixed with an equal volume of 21 sample buffer [0.125 M Tris– Cl (pH 6.8), 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.002% bromophenol blue]. Samples were heated in a boiling water bath for 5 min and then loaded onto a denaturing 6% polyacrylamide electrophoresis gel. After separation, proteins were electrophoretically transferred to a nitrocellulose membrane. The filters were first treated with Trisbuffered saline [50 mM Tris–Cl (pH 7.5), 0.15 M NaCl] with 0.05% Tween 20 (TBST) containing 3% nonfat milk. This was followed by incubation in 5 ml of TBST with 1% bovine serum albumin per milliliter and anti-Jun antibodies (No. SC44; Santa Cruz), anti-ER (No. SC543; Santa Cruz), and/or anti-a2 integrin antibody (Chemicon) at concentrations recommended by the manufacturers for 5 h at room temperature. The blots were subsequently washed in TBST and then incubated with the appropriate immunoglobulin peroxidase-conjugated antibodies (Amersham) for 1 h. After being washed in TBST, the blots were developed using the enhanced chemiluminescence Western immunoblotting system (Amersham). Analysis of the platelet fibrinogen receptor complex IIb–IIIa was performed similarly, except that 15 mg of total protein was used for electrophoresis and 2-mercaptoethanol was omitted from the sample buffer. Antibodies to Ilb–Illa were polyclonal rabbit serum (14) provided by Dr. Karen Knudsen of Lankenau Medical Research Center. Analysis of cellular differentiation. Erythroid differentiation was induced by adjusting logarithmically growing cells to either 0.36 mM ara C or 60 ng/ml activin A. After 5 days, the cells were stained with a 1/4 volume of benzidine solution (0.04% 3,3 *,5,5*-tetramethylbenzidine, 2% hydrogen peroxide, 12% acetic acid). Positive (blue-staining) cells were counted under a hemacytometer. A total of at least 200 cells were counted. Megakaryocytic differentiation was induced by adjusting cell cultures to 1008 M TPA for 2 days. Differentiation was assessed by cellular morphology, Wright–Giemsa staining, and Western analysis of the differentiation antigens Ilb–Illa and the a2 integrin. When estrogen was present in cultures, its concentration was 1 mM. AP-1 analysis. Measurement of AP-1 activity levels was assessed by transiently transfecting cells with pTRE-luc (15) (provided by Michael Karin of the University of California, San Diego) by the electroporation procedure described above. AP-1 activity induces luciferase activity from this construct by means of an artificial TPAresponse element (TRE) located in its promoter. After transfection, the cells were divided into two equal aliquots and one was adjusted to either 1008 M TPA or 1 mM estrogen. Subsequently, both were added to 10 ml of medium and incubated for 24 h. Cells were lysed in buffer containing 1% Triton X-100, and luciferase activity was measured on a Monolight 2010 luminometer (Analytical Luminescence Laboratory). All results presented are representative of at least three experiments.
RESULTS
Because randomly isolated sublines can vary from the parental line in the rate or extent of differentiation, there is a consequent difficulty in attributing the expression of an exogenous gene to a particular phenotypic change. These difficulties must be overcome by examining a large enough number of clones, with and without the exogenous gene, to collect statistically significant data. In cases in which partial effects are seen and experimental variation is problematic, the number of clones necessary to achieve such data can be large. We chose to avoid these difficulties by the use of two effective inducible systems in which the activity of the
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FIG. 1. Western Analysis of K562 transfectants expressing exogenous JunDN and DRaf-1:ER. Whole-cell protein extracts were prepared, subjected to electrophoresis and blotting, and analyzed with anti-Jun (A) or anti-ER (B) antibodies as described under Materials and Methods. Sources of extracts are as follows: (A) Lane 1, K562; lane 2, K562junDN (0tet); lane 3, K562junDN (/tet). (B) Lane 1, K562; lane 2, K562Draf-1:ER (0estrogen); lane 3, K562Draf-1:ER (/estrogen).
exogenous gene could be modulated within the same line. In one system the activity of c-Jun was modulated by the expression of a truncated c-jun gene, junDN , which encodes a product possessing the protein’s DNA binding and protein dimerization domains but lacking the transactivating domain. This construct has been shown to possess dominant-negative activity toward c-Jun or AP-1 (12). Inducible junDN expression was obtained by means of the tetracycline-responsive expression vector of Gossen and Bujard (12). In order to establish a subline of K562 expressing an inducible raf gene, we infected K562 cells with a retrovirus expressing the Draf1:ER gene of Samuels et al. (13). The active protein transduced by this construct is a hybrid protein containing sequences encoding a constitutively active Raf fused to the hormone binding domain of the estrogen receptor. The Raf sequences are a truncated c-Raf-1 similar to v-Raf and hence require no activation from upstream signal transduction. Raf activity in this construct is inhibited by the downstream ER sequences. However, in the presence of estrogen, the hormone binding sequences of ER assume a different conformation and cease to inhibit Raf activity. Figure 1A shows Western analysis of K562 and one of the K562junDN transfectants which were obtained. All transfectants expressed a 30-kDa dominant-negative protein (lane 2) which responded to tetracycline by decreased synthesis of JunDN to almost undetectable levels (lane 3). The antibody raised to C-terminal amino acids necessary to detect the N-terminal truncated protein did not detect endogenous c-Jun. Figure 1B shows Western analysis of K562Draf-1:ER with and without estrogen. The antibody raised to the hormone binding domain of the estrogen receptor detects a major protein band of approximately 67 kDa as originally re-
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ported by Samuels et al. (13). Also consistent with the original study, activation of the protein’s enzymatic activity by estrogen resulted in an increased transcriptional activation of the gene. Figure 2 shows the effect of the two exogenous genes on AP-1 activity. Cells were transiently transfected with the plasmid TRE-luc (15), which contains a luciferase reporter gene under the control of the collagenase promoter containing a single artificial TRE. Parental cells treated with TPA showed a 250-fold increase in AP-1 activity over untreated cells. There are two salient features of the figure. First, TPA treatment could be mimicked in K562Draf-1:ER by treatment with estrogen. Second, in K562junDN cells, junDN expression reduced AP-1 activity from 250-fold to 16-fold over cells not treated with TPA. Addition of tetracycline to the media restored a significant amount of AP-1 activity, eliminating the possibility that clonal variation incurred in the construction of K562junDN cell line could account for the response. We next used the K562junDN and K562Draf-1:ER clones to examine the effect of modulating AP-1 activity on induced differentiation. All K562junDN clones have a normal morphology and a growth rate which is unaltered by the level of junDN expression. However, the response to erythroid differentiation induced by either ara C or activin A was significantly affected by the presence of tetracycline. As seen in Fig. 3, ara C induced hemoglobin synthesis in the parental clone to the extent that approximately 65% of the cells stained blue with benzidine reagent. A lesser extent of hemoglobinization was seen in K562junDN cells in the presence of tet. This could be due either to a small amount
FIG. 2. AP-1 activity levels. Cell lines were transiently transfected with TRE-luc and analyzed for AP-1-induced luciferase activity as described under Materials and Methods. The relative luciferase activity is shown for the three cell lines under the indicated conditions.
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FIG. 3. Levels of erythroid differentiation induced by ara C or activin A as assessed by benzidine staining. Cells grown under the indicated conditions were stained with benzidine to assess hemoglobin levels as described under Materials and Methods. The percentage of benzidinepositive (blue-staining cells) under each condition is shown.
of JunDN expression in the presence of tet or to clonal variation between K562junDN and the parental line. However, the percentage of benzidine-positive cells was increased markedly in the K562junDN cells grown in the absence of tet. Comparable results were obtained when activin A was used as the inducer. Treatment of parental cells with this cytokine resulted in 30% of the cells differentiating. Again, the K562junDN clone exhibited a significantly higher level of differentiation with junDN induced over cells with junDN suppressed. Also shown in Fig. 3, treatment of K562Draf-1:ER cells with ara C resulted in 70% of the cells becoming benzidine positive, whereas the stimulation of Draf-1:ER activity by addition of 1 mM estrogen mimicked TPA treatment of K562 and thus almost completely inhibited this differentiation. Similar results were obtained when activin A was used as the inducer. All of these results are consistent with the concept that AP-1 activity inhibits erythroid differentiation. We next examined the effects of modulating AP-1 activity on TPA-induced megakaryocytic differentiation. Among the morphological changes induced by TPA treatment of K562 is a larger average cell volume and a spindle-shaped morphology among some of the cells in culture. The percentage of adherent cells also increases. Expression of junDN virtually eliminated the appearance of spindle-shaped cells induced by TPA treatment. When tet was added to the media of K562junDN , some of the spindle-shaped morphology inducible by TPA treatment was restored. However, the levels were not that of wild-type K562. As seen in Fig. 4, when analyzed cytochemically by Wright–Giemsa staining, untreated K562 cells show a relatively homogeneous population of blastlike cells with many cytoplasmic protrusions, a high (ú1) nuclear-to-cytoplasmic ratio, and round nuclei. Upon TPA-induced differentiation, the cell population progresses to one of more mature cells with a megakaryocyte morphology characterized by a lower (°1) nuclear-to-cytoplasmic ratio, a lobulated nucleus resulting from endomitosis,
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and a highly vacuolated cytoplasm. Also present are cells with a histiocytoid morphology. These are cells which appear spindle-shaped under light microscopy. They are characterized by a large acidophilic nucleus and lightly staining cytoplasm. No mitotic cells are evident. As expected, induction of AP-1 activity via induction of Raf activity in K562Draf-1:ER resulted in the cells assuming a more differentiated morphology. A quantitation of this change is presented in Table I. The extent of differentiation was not as great as that elicited by TPA treatment. Cell size increased similarly but cytoplasmic vacuolization was less extensive and some mitotic cells were still present. Consistent with the lack of spindle-shaped cells viewed under light microscopy, no histiocytic cells were apparent on Wright– Giemsa stains. As expected, expression of junDN decreased the above morphological changes induced by treatment with TPA. Cell size was still markedly increased and vacuolization was extensive. However, few histiocytic cells were evident. This is in contrast to K562junDN cells with junDN down-regulated by the presence of tet. Cytochemical analysis of cells in this case showed more abundant histiocytoid cells. In order to carry the analysis of TPA-induced differentiation further, the levels of megakaryocytic-associated proteins were measured. The platelet fibrinogen receptor complex antigen II–IIIa is a glycoprotein complex associated with platelets and megakaryocytes and is an early marker for differentiation along the megakaryocytic lineage (5). Fig. 5A shows Western blot analysis of IIb–IIIa in K562 and its derivatives using anti IIb–IIIa antibody (14). TPA treatment of K562 induced the synthesis of IIb–IIIa (lane 2), and as expected, induction of Raf activity in K562Draf-1:ER also induced the appearance of the IIb–IIIa antigen (lane 7). However, in contrast to the results obtained on cytochemical analysis, the inhibition of AP-1 by JunDN increased rather than decreased the level of IIb–IIIa expression (lanes 4 and 5). Similarly, the levels of the a2 integrin, which serves as a surface receptor for collagen on plate-
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FIG. 4. Wright – Giemsa staining of K562 cells and derivatives. The panel labeled K562 indicates untreated K562 cells. Immediately below are K562 cells treated with TPA. The middle two panels are K562junDN cells treated with TPA in the absence and the presence of tet. The right panels indicate K562Draf-1:ER cells without and with estrogen treatment.
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AP-1 ACTIVITY AND DIFFERENTIATION OF K562 CELLS TABLE I
Cytological Analysis of K562, K562junDN , and K567Draf-1:ER Before and After Treatment with TPA or Estrogena
a
Cell line
% Megakaryoblast
% Megakaryocyte
% Histiocytic
% Mitotic
K562 K562 / TPA K562junDN K562junDN/TPA K562junDN/tet/TPA K562Draf-1:ER0estrogen K562Draf-1:ER/estrogen
90 5 92 5 1 95 20
7 60 5 91 49 1 72
1 35 1 4 50 2 7
2 2
2 1
Cytochemical analysis was assessed by classification of cells into one of four categories based on criteria described in the text.
lets and other cells, were also examined. This megakaryocytic marker was also induced by Raf activity as in TPA treatment of K562 cells. Consistent with the experiments measuring IIb–IIIa levels, a2 integrin levels were also increased with junDN expression compared to the same cells with junDN down-regulated by tet (Fig. 5B). These results are in contrast to the results obtained by morphological and cytochemical analysis and indicate that while decreased AP-1 activity is associated with erythroid differentiation, induction of megakaryocytic differentiation is more complex than simply an increase in AP-1 activity. DISCUSSION
Experimental evidence suggesting that modulation of AP-1 activity is involved in erythroid differentiation dates back to work by Rovera et al. (16), who showed that erythroid differentiation of spontaneously differentiating sublines of F-MEL cells was abolished by TPA. This work was extended by Yamasaki et al. (17), who showed that TPA also inhibited hexamethylenebi-
FIG. 5. Western analysis of K562 cells and derivatives probed for IIb–IIIa expression (A) and a integin expression (B). Sources of extracts are as follows: (A) lane 1, untreated K562 cells; lane 2, K562 cells treated with TPA; lane 3, untreated K562junDN ; lane 4, K562junDN cells treated with TPA in the absence of tet; lane 5, K562junDN cells treated with TPA in the presence of tet; lane 6, K562Draf-1:ER cells in the absence of estrogen; lane 7, K562Draf1:ER cells in the presence of estrogen. (B) Extracts are the same as in (A).
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sacetamide-induced differentiation of F-MEL cells. The molecular basis for these observations lies in the mode of action of TPA. TPA is now known to be a potent inducer of AP-1 activity via activation of PKC (18). AP1 binds to the promoter sequence TGACTCA, also known as the TPA-response element (19), and thereby increases the expression of target genes responsible for most of the phenotypic changes elicited by TPA treatment. While increases in AP-1 activity are associated with decreased erythroid differentiation, there is also experimental evidence for the converse. Retinoic acid, an inducer of erythroid differentiation in one cell line (20), is an antagonist of AP-1 activity (21, 22). The mechanism of this inhibition probably involves a competition of these proteins for common coactivators (23). Additional evidence is provided by Francastel et al. (24), who studied a variant of F-MEL cells which was resistant to induced differentiation. They found that the resistance was associated with increased levels of cJun protein and that expression of anti-sense c-jun RNA could alleviate this inhibition. We have extended this association by experimentally modulating AP-1 activity by introduction of an inactive competitor to one of the AP-1 components, c-Jun. The truncated, dominant negative protein competes with the endogenous functional c-Jun for binding sites in c-Fos, thereby decreasing the concentrations of functional Fos/Jun dimers. This was seen in the initial experiments in which luciferase activity controlled by an AP-1-responsive promoter was significantly reduced by JunDN levels. Consistent with those previous studies cited above, downregulation of AP-1, while failing to initiate erythroid differentiation by itself, nevertheless, increased the extent of erythroid differentiation induced by two agents, ara C and activin A. To avoid any ambiguity in interpretation of the data, we eliminated the possibility of clonal variation by use of an effective inducible system. We found that the extent of induced erythroid differentiation could be reproducibly reversed within the same
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subline by adding or subtracting tet to the media of the cells. The involvement of AP-1 activity in the process of differentiation was further supported by experiments with a subclone of K562 expressing an inducible version of c-Raf. Cells with induced Raf activity showed elevated levels of AP-1, as assessed by luciferase activity under the control of a TRE element. This AP-1 activity, as in the case of TPA-induced AP-1 activity, not only inhibited erythroid differentiation induced by the two agents, but also elicited megakaryocytic differentiation. While this work was in progress, Whalen et al. (25) published similar results using constitutively active MAPK kinase expression vectors to induce megakaryocytic differentiation. The data discussed so far are consistent with the simple concept that decreases in AP-1 activity favor erythroid differentiation, whereas elevated levels favor megakaryocytic differentiation. However, this concept was not supported in experiments in which TPA treatment was used to induce megakaryocytic differentiation of K562 derivatives expressing JunDN . While JunDN expression inhibited the morphological changes induced by TPA treatment as expected, the gene product actually increased expression of two protein markers associated with the megakaryocytic phenotype. This observation indicates that control of lineage commitment is not as simple as modulating either the absolute level of AP-1 or the relative level of AP-1/NF-E2. This finding was perhaps presaged in previous work showing that NF-E2 is not exclusively an erythroid transcription factor (26, 27). While binding to NF-E2 sites in DNA appears to undergo a shift from NF-E2 to AP-1 binding upon TPA treatment, NF-E2 p45 mRNA is nevertheless increased (10). Similarly, knockout mice for the NF-E2 gene die of thrombocytopenia, while erythropoiesis is relatively normal (28). The control of lineage commitment thus appears to be a more complex and perhaps temporal mechanism. Differentiation of the cell culture along either lineage takes place over a period of several days. However, the expression and the activity of some key signal transduction components vary during the course of differentiation. For example, dimethyl sulfoxide or HMBA treatment of F-MEL cells induces an immediate increase in membrane-bound PKC activity (29–31). Depending on the cell line, this increased activity occurs by translocation of cytosolic PKC to the membrane or the activation of inactive membrane-bound PKC (32). In either case, this rise in activity is followed by a sustained decrease in total PKC activity occurring over the 5-day period of differentiation. Another time-dependent variable in differentiation events involves relative levels of the various AP-1 components. While the predominate members of AP-1 are often considered to be c-Fos and c-Jun, both are mem-
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bers of families of related proteins. The Fos family consists of c-Fos, Fos B, and Fra-1, while the Jun family consists of c-Jun, Jun-B, and Jun-D (33, 34). In some cell lines, Fos/Jun can induce the synthesis of Fra-1 (35). Serum growth factors stimulate transcription of fra-1 with delayed and protracted kinetics compared to c-fos and fos B induction (36, 37). Fra-1/Jun dimers bind with equal affinity to TREs but do not transactivate target gene expression as do Fos/Jun (38). Thus, Fos/Jun can, in some instances, induce the synthesis of their own inhibitor. To more fully characterize the molecular mechanisms of lineage commitment, the levels of NF-E2, Maf proteins, and AP-1 components will have to be followed throughout the course of differentiation. ACKNOWLEDGMENT The authors thank the people at Genetech for their generosity in supplying the activin A used in this study.
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