A Potential Role for Protein Kinase C-epsilon in Regulating Megakaryocytic Lineage Commitment*

Frederick K. RackeDagger , Dongyan Wang§, Zan ZaidiDagger , Joshua Kelley§, Jane Visvader, Jae-Won Soh||, and Adam N. Goldfarb§**

From the Dagger  Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, Maryland 21287, the § Department of Pathology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, the  Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria 3050, Australia, and the || Herbert Irving Comprehensive Cancer Center, Columbia College of Physicians and Surgeons, New York, New York 10027

Received for publication, June 16, 2000, and in revised form, September 29, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Multiple studies have shown that intracellular signal transduction by the protein kinase C (PKC) family participates in the initiation of megakaryocyte differentiation. In this study, multiple approaches addressed the functional contributions by specific PKC isozymes to megakaryocytic lineage commitment of two independent cell lines, K562 and human erythroleukemia (HEL). Pharmacologic profiles of induction and inhibition of megakaryocytic differentiation in both cell lines suggested a role for the calcium-independent novel PKCs, in particular PKC-epsilon . In transfection studies, the isolated variable domain of PKC-epsilon selectively blocked exogenous activation of the megakaryocyte-specific alpha IIb promoter. Constitutively active mutants of PKC-epsilon , but not of other PKC isozymes, cooperated with the transcription factor GATA-1 in the activation of the alpha IIb promoter. The functional cooperation between GATA-1 and PKC-epsilon displayed dependence on cellular milieu, as well as on the promoter context of GATA binding sites. In aggregate, the data suggest that PKC-epsilon specifically participates in megakaryocytic lineage commitment through functional cooperation with GATA-1 in the activation of megakaryocytic promoters.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A role for protein kinase C (PKC)1 signaling in megakaryocytic differentiation has been established by numerous experiments over the past two decades. In early studies, the PKC agonist phorbol diester selectively enhanced megakaryocyte colony formation by primary mouse bone marrow cells (1). More recent studies using primary human progenitors confirmed the promegakaryocytic effects of phorbol ester and showed such effects to be inhibitable by the PKC antagonists GF-109203X and Ro-31-8220 (2). In numerous cell line models of megakaryocytic differentiation, PKC activation induced an array of features including the following: cell cycle arrest, secretion of megakaryocytic cytokines, up-regulation of megakaryocytic surface antigens, cellular enlargement, polyploidization, development of proplatelet processes, and appearance of demarcation membranes (3, 4).

The PKC serine/threonine kinase family consists of at least 11 distinct isozymes organized into three subgroups, based on biochemical, pharmacologic, and structural properties (5). The classical or conventional PKCs (cPKCs) require diacylglycerol and Ca2+ for activation and consist of the alpha , beta I, beta II, and gamma  isozymes. The novel PKCs (nPKCs) require only diacylglycerol for activation and consist of the delta , epsilon , theta , eta , and µ isozymes. The atypical PKCs lack responsiveness to diacylglycerol and Ca2+ and consist of the lambda  and zeta  isozymes. Striking functional differences exist among PKC isozymes, with divergent functions noted even for factors with high structural homology (6-8)

PKC signaling may influence megakaryocytic differentiation through several isozymes. In K562 cells, PKC-mediated, sustained activation of the Raf-MEK-ERK signaling pathway is necessary for initiation of megakaryocytic differentiation (9, 10). Multiple PKC isozymes, in particular alpha , beta I, eta , and delta , possess the capacity to activate the Raf-MEK-ERK pathway (11, 12). However, activation of the Raf-MEK-ERK pathway appears not to be required for megakaryocytic differentiation of primary progenitor cells, suggesting that PKC signaling plays an additional role in megakaryopoiesis independent of ERK activation (13).

To examine the contribution of specific PKC isozymes to megakaryocyte differentiation, we initially employed isozyme-selective pharmacologic agents in two independent cell line models of megakaryocytic differentiation, K562 and HEL. GF-109203X, an inhibitor of cPKCs and nPKCs but not of atypical PKCs, potently blocked megakaryocytic induction in both cell lines. By contrast, Gö 6976, an inhibitor only of cPKCs (14), failed to block megakaryocytic differentiation, suggesting a specific requirement for nPKC signaling. For both cell lines, the PKC-epsilon -selective agonist, ingenol 3,20-dibenzoate (IDB) (15-17) induced megakaryocytic differentiation, as well as selective nuclear translocation of PKC-epsilon .

In transfection assays, the isolated variable domain of PKC-epsilon , but not that of PKC-alpha , completely blocked exogenous activation of the megakaryocytic alpha IIb promoter. Constitutively active mutants of PKC-epsilon activated the alpha IIb promoter 3-6-fold. We also addressed whether PKC-epsilon signaling influenced the function of GATA-1, a transcription factor known to play a critical role in megakaryopoiesis and in activation of the alpha IIb promoter (18-20). Indeed, GATA-1 and constitutively active PKC-epsilon showed synergistic activation of the alpha IIb promoter. Notably, the ability to synergize with GATA-1 distinguished PKC-epsilon from other PKC isozymes, depended on cellular milieu, and depended on the context of GATA binding sites within the promoter.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- K562 and HEL, obtained from the ATCC, were grown in RPMI 1640 with 10% fetal bovine serum at 37 °C, 5% CO2. C3H10T1/2, obtained from ATCC, was grown in Dulbecco's modified Eagle's medium with 10% neonatal calf serum at 37 °C, 5% CO2. HEK-293T, provided by Dr. Kevin Lynch (Department of Pharmacology, University of Virginia School of Medicine), was grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum at 37 °C, 5% CO2. All experiments using K562 and HEL employed mid-log phase cells at a density of 0.5-1.0 × 106 cells/ml. Conditioned media was obtained as described previously by 72 h of treatment of either K562 or HEL cells with 25 nM 12-O-tetradecanoylphorbol-13-ester (Sigma) followed by harvesting and dialysis of supernatant (9).

For megakaryocytic induction, cells were resuspended in conditioned media and incubated at 37 °C, 5% CO2 for 1-3 days, as indicated. The compounds GF-109203X, Gö 6976, and IDB were purchased from LC Laboratories. The PKC inhibitors GF-109203X and Gö 6976 were added, at indicated concentrations, to conditioned media at the initiation of megakaryocytic induction.

Flow Cytometry-- Staining of cells for surface CD41 employed the fluorescein isothiocyanate-conjugated antibody PLT1-FITC (Coulter) at 25 µg/ml. Staining of cells for surface glycophorin A employed the phycoerythrin-conjugated antibody GA-R2-PE (Pharmingen) at 10 µg/ml. Appropriate fluorochrome-conjugated, isotype-matched antibody controls were used at concentrations identical to the corresponding experimental antibodies. Flow cytometric analysis was performed on a FACScan system utilizing Lysys II software (Becton Dickinson).

Immunofluorescent Staining for PKC-- Treated cells were cytospun onto glass slides and fixed for 2 min in ice-cold methanol followed by 2 min in ice-cold acetone. After blocking for 30 min at room temperature with 1% normal goat serum in phosphate-buffered saline, primary antibodies in 0.1% normal goat serum/phosphate-buffered saline were applied for 1 h at room temperature. Murine monoclonal antibodies to PKC-epsilon (Santa Cruz Biotechnology, Santa Cruz, CA) and to PKC-alpha (Transduction Laboratories, Lexington, KY) were used at 200 ng/ml and at 1.25 µg/ml, respectively. Control murine antibody (NOR 3.2; BIOSOURCE International) was used at 1 µg/ml. Secondary antibody, consisting of phycoerythrin-conjugated goat anti-mouse (Tago, Inc.) diluted 1:100 in 0.1% normal goat serum/phosphate-buffered saline, was applied for 30 min at room temperature. For nuclear visualization, 4,6-diamidino-2-phenylindole was included in the coverslip mounting medium. Cells were visualized by confocal laser scanning fluorescence microscopy on a Zeiss LSM 410 (Jena, Germany) using Zeiss LSM analysis software.

Plasmid Constructs-- The alpha IIb-luciferase reporter constructs were made by polymerase chain reaction amplification of bases -598 to +32, bases -98 to +32, or bases -348 to +32 from a human alpha IIb promoter fragment kindly provided by Dr. Samuel Santoro (21). The polymerase chain reaction products were co-digested with XhoI plus HindIII and ligated into the corresponding sites of pGL3-Basic (Promega). The beta -galactosidase expression vector consisted of pCMVbeta (CLONTECH). The GATA-1 expression vector employed the EF-1-alpha -neo expression plasmid and has been previously described (22).

Mammalian expression of a full-length constitutively active (CA) mutant of PKC-epsilon employed SRD-epsilon -K155A/R156A/A159E (AE3), kindly provided by Dr. S. Ohno (23). Expression of a full-length CA mutant of PKC-delta employed SRD-delta R144/145A (DRA), also kindly provided by Dr. S. Ohno (23). Expression of a full-length CA mutant of PKC-alpha employed pRc-CMV-PKC-alpha A25E, kindly provided by Dr. Gottfried Baier (24). Expression of a full-length CA mutant of PKC-theta employed pRc/CMV-PKC-theta R145I/R146W, kindly provided by Dr. J. Anthony Ware (25). Mammalian expression constructs for HA-epitope-tagged, isolated PKC catalytic and regulatory domains have been previously described (26). In brief, fragments encoding the catalytic domains of alpha  (amino acids 326-672) and epsilon  (amino acids 395-737) were ligated into the pHANE vector. The fragment encoding the catalytic domain of delta  (amino acids 334-674) was ligated into the pHACE vector. Fragments encoding the variable domains of alpha  (amino acids 2-325) and epsilon  (amino acids 2-394) were ligated into the pHANE vector.

Transfections and Reporter Assays-- Transfection of K562 and C3H10T1/2 cells employed the liposomal reagent DOTAP (Roche Molecular Biochemicals) using ~6 µg of DNA in 200 µl of HBS (20 mM HEPES, 150 mM NaCl, pH 7.4) combined with 30 µl of DOTAP in 200 µl HBS. The DNA/DOTAP mixture was incubated for 10 min at room temperature and was then added dropwise to 1. 6 × 106 K562 cells in 2 ml of RPMI 1640 with 5% fetal bovine serum. For C3H10T1/2 cell transfections, the DNA/DOTAP mixture was added to ~60% confluent cells in 6-well plates. Transfection of HEK-293T cells was performed as described for C3H10T1/2 cells except that 4 µg of DNA in 50 µl of HBS was combined with 20 µl of DOTAP in 100 µl of HBS.

After overnight incubation, cells were changed to fresh complete medium. Cells were subsequently incubated 24 h prior to harvesting for luciferase and beta -galactosidase assays. Luciferase assays were performed using the commercial luciferase assay system (Promega), and beta -galactosidase assays were performed using the O-nitrophenyl beta -D-galactopyranoside (Sigma) colorimetric substrate. All transfections were performed at least in triplicate, and all luciferase values were normalized according to beta -galactosidase readings.

Immunoblot Assays-- K562, C3H10T1/2, and HEK-293T cells were transfected and harvested as for the luciferase and beta -galactosidase assays, except that whole cell lysates were prepared by resuspending cells in 1× SDS polyacrylamide gel electrophoresis loading buffer. Samples were resolved by SDS polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Equivalent lane loading was confirmed by Ponceau staining of membranes. Probing of membranes was carried out as described previously (27). For detection of HA-tagged PKC regulatory and catalytic domains, the primary antibody consisted of 12CA5, a murine monoclonal directed to the HA epitope tag, employed as ascites fluid diluted 1:2000. The secondary antibody consisted of peroxidase-conjugated goat anti-mouse (Sigma) used at a dilution of 1:3000. For detection of GATA-1, the primary antibody consisted of the rat monoclonal N6 (Santa Cruz Biotechnology) at a final concentration of 0.4 µg/ml. The secondary antibody consisted of peroxidase-conjugated goat anti-rat (Sigma) used at a dilution of 1:5000. Signal detection employed enhanced chemiluminescence.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Pharmacologic Implication of nPKC in Megakaryocytic Differentiation-- Previous work in our laboratory indicated that sustained activation of the Raf-MEK-ERK pathway in the K562 hematopoietic cell line resulted in production of autocrine factors promoting megakaryocytic maturation (9). To identify signaling pathways triggered by such autocrine factors, we analyzed the effects of pharmacologic inhibitors on megakaryocytic induction in two independent cell lines, K562 and HEL. The induction stimulus consisted of conditioned media from 12-O-tetradecanoylphorbol-13-ester-treated HEL cells, which show identical activity to that previously reported for K562 cells (9).

The only compound in our screening that potently and specifically blocked megakaryocytic induction by conditioned media was GF-109203X. GF-109203X inhibits both cPKC and nPKC isozymes, as well as pp90rsk2 (14, 28). Therefore, parallel experiments were carried out using the compound Gö 6976, known to inhibit cPKCs and pp90rsk2 but not nPKCs (14, 28). As shown in the flow cytometric profiles in Fig. 1, conditioned media alone induced up-regulation of the megakaryocyte surface antigen CD41 and down-regulation of the erythroid surface antigen glycophorin A. Whereas GF-109203X completely blocked both responses, Gö 6976 at similar doses showed no inhibition of either response.



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Fig. 1.   Effects of the PKC inhibitory compounds, GF-109203X and Gö 6976, on megakaryocytic differentiation of the K562 and HEL cell lines. Cells were exposed for 72 h to conditioned medium either alone or containing the indicated agents at a concentration of 3 µM. Cells were stained with the indicated fluorochrome-conjugated antibodies and analyzed by flow cytometry. The hollow profiles represent uninduced cells, and the shaded profiles represent conditioned medium-induced cells. This experiment was performed on three separate occasions, each yielding similar results. GPA, glycophorin A.

Analysis of cellular morphology supported the flow cytometric results in Fig. 1. In particular, HEL cells exposed to conditioned media undergo spreading and enlargement as part of their megakaryocytic differentiation. As shown in Fig. 2, the morphologic changes induced in HEL cells by conditioned media were abrogated by GF-109203X. By contrast, Gö 6976 strikingly enhanced the cellular spreading and enlargement induced by conditioned media. These data confirm that biologically active doses of Gö 6976 acted to enhance rather than inhibit features of megakaryocytic differentiation.



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Fig. 2.   Effects of the PKC inhibitors, GF-109203X and Gö 6976, on the morphologic differentiation of HEL cells. Cells were induced by treatment for 48 h with conditioned medium alone or with the indicated agents at 10 µM. Cells were visualized by phase contrast microscopy at a magnification of × 200. This experiment was performed on three separate occasions, each yielding similar results.

In an alternative approach, the isozyme-selective PKC agonist IDB was applied directly to K562 and HEL cells in standard growth media. Multiple previous studies have indicated that IDB is a selective activator of nPKCs, particularly PKC-epsilon (15-17). As shown in Fig. 3, IDB caused CD41 up-regulation and glycophorin A down-regulation in K562 and HEL cells. In addition, HEL cells treated with IDB manifested the standard morphologic changes seen with megakaryocytic induction (not shown). Immunofluorescent staining (Fig. 4) showed that treatment of K562 cells with IDB induces rapid nuclear translocation of PKC-epsilon but no change in the subcellular localization of PKC-alpha . Rapid nuclear translocation of PKC-epsilon was also observed in HEL cells treated with IDB (data not shown). Thus, both agonists and antagonists implicate nPKC, in particular PKC-epsilon , in the induction of megakaryocytic differentiation.



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Fig. 3.   Induction of megakaryocytic differentiation with the nPKC-selective agonist IDB. Cells were treated for 4 days with 100 nM IDB prior to analysis by flow cytometry. Hollow profiles indicate control cells treated with Me2SO carrier, and shaded profiles indicate cells treated with IDB. This experiment was performed on three separate occasions, each yielding similar results. GPA, glycophorin A.



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Fig. 4.   Immunofluorescent subcellular localization of PKC-epsilon and PKC-alpha in K562 cells, untreated and treated with IDB. Cells were treated for 10 min at 37 °C with or without 100 nM IDB. Following stimulation, cells were fixed and stained as described under "Materials and Methods." The images represent the following: Left column, blue staining indicates 4,6-diamidino-2-phenylindole staining of nuclei. Middle column, anti-PKC immunoreactivity indicated by red staining. Right column, composite image superimposing left and middle columns demonstrating nuclear localization of PKC-epsilon following IDB treatment. This experiment was performed on three separate occasions, each yielding similar results.

Blockade of Megakaryocytic Promoter Activation by the Regulatory Domain of PKC-epsilon -- The amino-terminal regulatory sequences of PKCs, when expressed as isolated fragments, function as dominant-negative PKC inhibitors (29, 30). Accordingly, we examined whether transfection of isolated regulatory domains from either PKC-alpha or PKC-epsilon could interfere with exogenous activation of the megakaryocyte-specific alpha IIb-598 promoter (31). In cells transfected with control vector, 24 h of conditioned media caused an ~14-fold up-regulation of the alpha IIb-598-luciferase reporter activity (Fig. 5A). Expression in cells of the isolated alpha  regulatory fragment (alpha -Reg) minimally inhibited conditioned media activation of alpha IIb-598-luciferase. In striking contrast, expression in cells of the isolated epsilon  regulatory fragment (epsilon -Reg) almost completely eliminated responsiveness to the conditioned media stimulus. Immunoblotting showed similar expression levels of the HA-epitope-tagged alpha  and epsilon  regulatory domains in HEK-293T transfectants (Fig. 5B).



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Fig. 5.   A, the effects of PKC regulatory domains (Reg) on exogenous activation of the megakaryocytic alpha IIb promoter. Control vector or expression vectors for the alpha  or epsilon  regulatory domain (3 µg) were cotransfected with the alpha IIb-598-luciferase reporter plasmid (1 µg), as well as a beta -galactosidase expression vector (1 µg), into K562 cells. Cells were then subjected to 24 h of induction with conditioned media. Results are shown as -fold increase in luciferase activity relative to uninduced cells transfected with control vector. All results are normalized by beta -galactosidase activity and represent the mean of three experiments ± S.E. B, immunoblot analysis of HEK-293T transfectants showing expression of the HA-epitope-tagged PKC regulatory domains. Positions of molecular mass markers are indicated to the left of the blot; from top to bottom they are as follows: 97, 66, 55, 43, 40, and 31 kDa. IB, immunoblot.

Specific Cooperation of PKC-epsilon with GATA-1 in Activation of a Megakaryocytic Promoter-- We next tested whether constitutively active PKC mutants could activate the alpha IIb megakaryocytic promoter. Fig. 6A demonstrates the similar results obtained with two different types of constitutively active mutants, inhibitory domain point mutants (CA mutants in left graph), and isolated PKC catalytic domains (CAT mutants in right graph). The inhibitory domain point mutants consist of full-length PKCs with point mutations in the autoinhibitory regulatory domains (23-25). The isolated PKC CAT completely lack regulatory domains, which have also been implicated in Ca2+ and lipid binding, interaction with RACKs, and kinase-independent signaling (26). Using the megakaryocyte-specific alpha IIb-598 reporter, we tested the effects of PKC isozymes alone and in conjunction with GATA-1, a known positive regulator of the alpha IIb promoter (18, 19). As shown in Fig. 6A, constitutively active PKC-epsilon mutants alone modestly activated the alpha IIb promoter (3-6-fold) but demonstrated clear functional cooperation with GATA-1. In fact, coexpression of GATA-1 with either CA or CAT mutants of epsilon  led to levels of reporter activation analogous to those obtained with conditioned media induction. Functional cooperation with GATA-1 was clearly isozyme-restricted in that constitutively active mutants of alpha , delta , and theta  all failed to augment GATA-1-mediated alpha IIb activation. In fact, delta  caused a 2-3-fold inhibition of GATA-1 activation. Immunoblot analysis demonstrated equivalent levels of GATA-1 expression in all transfectants, indicating that the differential effects of the PKC isozymes were not because of differences in GATA-1 levels (Fig. 6B). In addition, immunoblot demonstrated analogous expression levels of the HA-epitope-tagged epsilon , alpha , and delta  catalytic domains in HEK-293T cells (Fig. 6C).



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Fig. 6.   Functional cooperation of PKC-epsilon with GATA-1 in the activation of the megakaryocytic alpha IIb promoter. A, activity of constitutively active PKC mutants ± GATA-1. CA-epsilon represents PKC-epsilon K155A/R156A/A159E; CA-delta represents PKC-delta R144/145A; CA-theta represents PKC-theta R145I/R146W; and CA-alpha represents PKC-alpha A25E. Isolated PKC catalytic domains are designated as CAT. B, immunoblot (IB) analysis of transfectants for GATA-1 expression. Positions of molecular mass markers are indicated to the left of the blot; from top to bottom they are as follows: 97, 66, 55, 43, 40, and 31 kDa). C, immunoblot analysis of HEK-293T transfectants showing expression of the HA-epitope-tagged PKC catalytic domains. Molecular mass markers are designated as in B. K562 cells were transfected with 2 µg of PKC expression vector (or parent vector), 2 µg of GATA-1 expression vector (or parent vector), 2 µg of the alpha IIb-598-luciferase reporter plasmid, and 0.5 µg of beta -galactosidase expression vector. Results are shown as -fold increase in luciferase activity relative to cells transfected with reporter plasmid plus control vectors. All results are normalized by beta -galactosidase activity and represent the mean of three experiments ± S.E.

Cellular Context and Promoter Context Influence PKC-epsilon /GATA-1 Cooperativity-- To determine whether functional interaction between PKC-epsilon and GATA-1 occurred also in non-hematopoietic cells, cotransfections were carried out in C3H10T1/2 fibroblasts rather than in K562 hematopoietic cells. The full-length CA epsilon  mutant, as well as the isolated epsilon  CAT, failed to augment GATA-1-mediated alpha IIb activation in C3H10T1/2 fibroblasts (Fig. 7A). Immunoblotting demonstrated equivalent expression of GATA-1 in all of the transfectants (Fig. 7B). Thus, the functional interaction of PKC-epsilon with GATA-1 clearly depends on the cell type employed for transfection.



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Fig. 7.   Influences of cellular milieu and promoter context on PKC-epsilon /GATA-1 cooperativity. A, C3H10T1/2 fibroblasts were transfected with 2 µg of GATA-1 expression vector (or parent vector), 2 µg of PKC-epsilon expression vector (or parent vector), 2 µg of the alpha IIb-598 reporter plasmid, and 0.5 µg of beta -galactosidase expression vector. CA-epsilon represents the constitutively active regulatory domain point mutant PKC-epsilon K155A/R156A/A159E. epsilon -CAT represents the constitutively active isolated PKC-epsilon catalytic domain. B, immunoblot (IB) analysis of transfectants for GATA-1 expression. Positions of molecular mass markers are indicated to the left of the blot; from top to bottom they are as follows: 97, 66, 55, 43, 40, and 31 kDa). C, K562 cells were transfected with the alpha IIb-598 reporter plasmid or with the 5' truncated reporter plasmids alpha IIb-98 and alpha IIb-348. In addition, the cells received the constitutively active mutants of PKC-epsilon (versus parent vector) and wild type GATA-1 (or parent vector). Results are shown as -fold increase in luciferase activity relative to cells transfected with reporter plasmid plus control vectors. All results are normalized by beta -galactosidase activity and represent the mean of three experiments ± S.E.

The alpha IIb promoter contains multiple GATA binding sites, including a functional site within the promoter-proximal -98 fragment (18, 19). To determine whether specific promoter regions were required for PKC-epsilon /GATA-1 cooperativity, 5' truncated reporter constructs, alpha IIb-98 and alpha IIb-348, were compared with alpha IIb-598 for responsiveness to PKC-epsilon  ± GATA-1 in K562 cells. Surprisingly, the alpha IIb-98 and alpha IIb-348 reporters showed full activation by GATA-1 alone but no evidence of augmentation by PKC-epsilon (Fig. 7C). Thus, the functional GATA binding sites in the alpha IIb -348 to +32 fragment were insufficient to mediate PKC-epsilon /GATA-1 cooperativity.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Involvement of PKC signaling in hematopoietic lineage commitment decisions has been well documented. In progenitors transformed by the E26 avian leukemia virus, thresholds of PKC activity correlated with cell fate determinations as follows: (a) no kinase activity was associated with undifferentiated cells; (b) low activity was associated with myelomonocytic differentiation; and (c) high activity was associated with eosinophil differentiation (32). In primary, bipotential granulocyte macrophage colony-forming cells, activation of PKC-alpha induced commitment to the macrophage lineage (33). Similarly, our data suggest that signaling via PKC-epsilon may promote megakaryocytic lineage commitment of the bipotential BFU-E/MK progenitor, a cell with capability for either erythroid or megakaryocytic differentiation (34). Previous studies have notably shown that PKC-epsilon undergoes down-regulation during erythroid differentiation and that inhibition of PKC-epsilon specifically enhances erythroid differentiation (35, 36).

Mechanisms by which PKC-epsilon signaling might contribute to the activation of the megakaryocytic alpha IIb promoter remain unclear. Earlier studies with isolated catalytic domains have shown PKC-epsilon to activate multiple pathways that converge on the serum-response element of the c-fos promoter, c-Raf-MEK1-ERK, MEK kinase 1-stress-activated protein kinase kinase-c-Jun NH2-terminal kinase, and rhoA (26). However, those studies showed equivalent activation of the various pathways by the PKC-alpha catalytic domain. Our results, by contrast, show no activation of the alpha IIb promoter by the PKC-alpha catalytic domain. The rapid nuclear translocation of PKC-epsilon observed with megakaryocytic induction by IDB (Fig. 4) raises the possibility that PKC-epsilon itself might act directly upon critical nuclear substrates.

The functional cooperation of PKC-epsilon with GATA-1 raises a number of mechanistic possibilities. One scenario is that PKC-epsilon signaling targets a transcriptional complex containing GATA-1 and enhances GATA-1 function by phosphorylation of one of the members of this complex, such as GATA-1 itself or the cofactor friend of GATA-1. The absence of functional interaction in C3H10T1/2 cells argues against direct phosphorylation of GATA-1 by PKC-epsilon as a sufficient mechanism. This scenario also fails to account for the dependence of PKC-epsilon signaling on promoter context, as illustrated in Fig. 7C. Accordingly another possibility is that PKC-epsilon signaling targets GATA-1 complexes binding to specific regions of the alpha IIb promoter. A recent study employing embryonic stem cell hematopoiesis has defined within the human alpha IIb promoter a 200-base pair critical enhancer region, -398 to -598, that is necessary and sufficient for megakaryocyte-specific transgene expression (31). Interestingly, our data indicate that a similar region (from -348 to -598 of the human alpha IIb promoter) is required for responsiveness to PKC-epsilon signaling. Future studies will attempt to correlate PKC-epsilon response elements within the alpha IIb promoter with megakaryocyte-specific enhancer function.

A major question in the molecular characterization of hematopoietic lineage commitment is how two lineages with highly similar arrays of transcription factors can show non-overlapping, indeed mutually exclusive, patterns of gene expression. Erythroid and megakaryocytic cells share expression of the highly restricted factors GATA-1, GATA-2, Lmo2, NF-E2, friend of GATA-1, and SCL/tal. Most striking among these factors is GATA-1, which dominantly activates erythroid genes only in erythroblasts and dominantly activates megakaryocytic genes only in megakaryocytes. Our current data raise the possibility that isozyme-specific signaling by PKC may modify GATA function in accordance with promoter context. In particular, PKC-epsilon signaling might specifically augment GATA-1 function in the context of megakaryocytic promoters, thereby redirecting the entire transcriptional program of a cell from erythroid to megakaryocytic.


    ACKNOWLEDGEMENTS

We thank Kristine Lewandowska for excellent technical assistance in the early phases of this project. For generously providing plasmid constructs, thanks go to Drs. Samuel Santoro, Gottfried Baier, J. Anthony Ware, and S. Ohno. For helpful discussions and support, thanks go to Drs. Chi V. Dang, Isa Hussaini, and Julianne J. Sando.


    FOOTNOTES

* This work was supported in part by the Concern II Foundation (to A. N. G.), Public Health Service Grant CA-72704 from the NCI, National Institutes of Health (to A. N. G.), the Johns Hopkins Solo Cup Clinician Scientist Award (to F. K. R.), and Public Health Service Grant HL-04017 from the NHLBI, National Institutes of Health (to F. K. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed: Dept. of Pathology, HSC Box 800214, University of Virginia Health Sciences Center, Charlottesville, VA, 22908. Tel.: 804-982-0593; Fax: 804-924-8060; E-mail: ang3x@virginia.edu.

Published, JBC Papers in Press, October 12, 2000, DOI 10.1074/jbc.M005236200


    ABBREVIATIONS

The abbreviations used are: PKC(s), protein kinase C(s); cPKC(s), classical or conventional PKC(s); nPKC(s), novel PKC(s); MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ERK, extracellular signal-regulated kinase; IDB, ingenol 3,20-dibenzoate; HEK, human embryonic kidney; CA, constitutively active; HA, hemagglutinin; DOTAP, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium salts; CAT, catalytic domains; HEL, human erythroleukemia.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


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