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Cell Growth & Differentiation Vol. 12, 435-445, August 2001
© 2001 American Association for Cancer Research

Inducible Expression of the Megakarocyte-specific Gene Glycoprotein IX Is Mediated through an Ets Binding Site and Involves Upstream Activation of Extracellular Signal-regulated Kinase1

Michael Eisbacher, Levon M. Khachigian2, Thet Hta Khin, Melissa L. Holmes and Beng H. Chong2,, 3

The Centre for Thrombosis and Vascular Research, School of Pathology, University of New South Wales and Department of Medicine, St. George Hospital, Sydney, NSW 2217 Australia


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Glycoprotein IX is a megakaryocyte-specific gene crucial for adequate and functional expression of the Glycoprotein Ib-IX complex. This study used phorbol 12-myristate 13-acetate (PMA) and thrombopoietin (TPO)-induced differentiation of Dami and UT-7 cells, respectively, to investigate the regulation of inducible Glycoprotein IX expression during megakaryocyte differentiation. PMA and TPO were able to modulate GPIX expression at mRNA and protein levels. Transient transfection studies using nested 5'-deletions and mutations of the GPIX promoter demonstrated the absolute requirement of an inverted Ets site 5'-ACTTCCT-3' for inducible reporter gene expression. The upstream signaling events associated with PMA and TPO-inducible expression of GPIX were also investigated. The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase inhibitor PD98059 inhibited both PMA and TPO-inducible reporter activity in a dose-dependent manner, whereas inhibition of p38/MAPK had no significant effect. The protein kinase C inhibitor GF109203X failed to inhibit TPO-activation of the GPIX promoter in UT-7 cells. This study demonstrates that inducible expression in response to either PMA or TPO is mediated through the Ets site in the proximal promoter of GPIX and is dependent upon the upstream activation of MAPK/extracellular signal-regulated kinase.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Hematopoiesis is a complex process involving the interplay of cytokines, growth factors, and cell adhesion events, ultimately resulting in a specific pattern of gene expression, which in turn commits cells to a specific lineage (1, 2, 3) . The process of MK4 differentiation involves an increase in cell size, endomitotic division, and the formation of a demarcation membrane, culminating in the release of functional platelets (4) . Central to megakaryopoiesis is the expression of a restricted set of MK specific genes (5) . Some of these genes include: GPIIb, GPIb{alpha}, GPIX, GPV, platelet factor 4, and the gene for the TPO receptor c-Mpl. Studies (6, 7, 8, 9, 10, 11, 12) on the promoter regions of these genes have shown they exhibit several common features, including the lack of a TATA box and the existence of binding sites for GATA and Ets family members.

GPIX is a relatively small surface protein that comprises part of the GPIb-IX complex (13) . The GPIb-IX complex contains the binding site for vWf and is crucial for the initial adhesion of platelets to the vessel wall during bleeding (14) . GPIX is believed to play an essential role in stabilizing the formation of the GPIb-IX complex (15) . Failure to express the GPIX subunit results in inadequate and dysfunctional assembly of the complex on the cell surface (16) . GPIb-IX deficiency is manifested clinically by a congenital disorder known as Bernard Soulier Syndrome. This rare disorder is characterized by bleeding and abnormal "giant" platelets (17 , 18) . The existence of morphologically abnormal MKs and platelets in Bernard Soulier Syndrome (19 , 20) suggests that expression of the GPIb-IX complex is an important step during normal terminal differentiation of MKs. Moreover, recent evidence (21) suggests that expression of the GPIb-IX complex may play a role in regulating megakaryopoiesis, firstly by inducing expression of the cyclin-dependent kinase inhibitor p21, which has been implicated in polyploidization, and secondly by inducing G1 cell cycle arrest. Expression of GPIX, therefore, is an important event during MK development.

The Ets family of transcription factors consists of over 40 members that are expressed over a wide variety of species (22) . Members of the Ets family contain a common Ets binding domain that interacts with DNA sequences related to 5'-C/AGGAA/T-3' (23) . Ets transcription factors have been implicated in cellular development. The Ets family members Ets-1, Fli-1, and PU.1/Spi-1 are present in megakaryocytic cell lines, MKs, and platelets, and evidence for their importance in MK differentiation is beginning to accumulate (6 , 24) ; e.g., the Ets factor PU.1/Spi-1 has been shown to be important for TPO-induced expression of the GPIIb gene in UT-7 cells (25) . Second, overexpression of Fli-1/ErgB in K562 cells results in an increased MK phenotype and induces changes similar to those observed when the cells are treated with the differentiating agent 12-O-tetradecanoylphorbol-13-acetate (26) . A number of Ets family members are able to transactivate the promoters of GPIIb, GPIb{alpha}, GPIX, vWf, and the TPO receptor c-Mpl when cotransfected into cell lines with either nonhematopoietic or megakaryocytic features (6 , 24 , 25 , 27) . Furthermore, it was demonstrated recently (28) that inactivation of the Fli-1 gene in mice resulted in embryonic death in homozygotes because of hemorrhage.

The cytokine TPO is the major regulator of proliferation and differentiation of MK progenitors during hematopoiesis. Binding of TPO to its receptor c-Mpl results in the activation of a number of different signaling cascades within the cell, which include the Jak-Stat, Ras/MAPK, and PKC pathways (29, 30, 31, 32, 33, 34) . A number of investigators (35, 36, 37, 38) have demonstrated the importance of the Ras/MAPK pathway for megakaryocytic differentiation and, in particular, for expression of the MK-specific gene GPIIb, an early marker of MKs. However, important links between activation of these signaling pathways by TPO and expression of genes such as those in the GPIb-IX complex during terminal differentiation of MKs remain to be identified.

In this study, we have characterized the profile of GPIX expression in MK cell lines in response to both PMA and TPO to identify candidate transcriptional regulators and signaling pathways important for GPIX expression during terminal differentiation. This work demonstrates the importance of the Ets binding site in the proximal promoter for both basal and inducible expression of GPIX. Moreover, we provide evidence that inducible GPIX expression in response to either PMA or TPO is dependent upon the upstream activation of ERK.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
PMA Induction of GPIX mRNA and Surface Expression in Dami Cells Is Attributable to New Protein Synthesis.
PMA has previously been widely used as a model agonist to promote differentiation of cell lines with megakaryocytic features. More specifically, PMA is able to up-regulate the expression of MK-specific genes such as GPIIb and PF4 (37 , 39) . In this present study, Northern blot analysis was carried out initially to test whether PMA could induce the expression of the MK-specific gene GPIX in Dami cells to characterize the profile of GPIX mRNA expression during differentiation. Total RNA was harvested from growth-arrested Dami cells exposed to 100 nmol/liter PMA at various time intervals. Incubation with PMA induced GPIX mRNA expression within 12 h, which was further increased at 24 and 48 h (Fig. 1)Citation . Because of the somewhat delayed response, we repeated the experiment in the presence of the protein synthesis inhibitor cycloheximide. Preincubation with cycloheximide completely abrogated the ability of PMA to induce GPIX expression, indicating the requirement of new protein synthesis. PMA-inducible expression of GPIX mRNA was also dose-dependent. A concentration as low as 5 nmol/liter was able to induce GPIX mRNA expression, with a maximal response observed at 100 nmol/liter (data not shown). Membranes were stripped and probed for the housekeeping gene GAPDH to demonstrate equal loading of RNA in each lane.



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Fig. 1. PMA induces GPIX mRNA expression in Dami cells. The ability of PMA to modulate GPIX mRNA expression in Dami cells was initially evaluated by Northern blotting. Dami cells were growth-arrested overnight and incubated with PMA (100 nmol/liter). Total RNA was harvested at times indicated and subject to Northern blotting. Membranes were rehybridized with [32P]GAPDH cDNA to demonstrate the amount of RNA loaded. Results are representative of three independent experiments.

 
It is well established that PMA can stimulate the surface expression of MK markers such as GPIb{alpha}, {alpha}IIbß3, and {alpha}2ß1 in cell lines with megakaryocytic features (36 , 37 , 40) ; however, to our knowledge, there are no reports for GPIX. Dami cells were growth-arrested overnight and then incubated with either 100 nmol/liter PMA or vehicle alone (DMSO). The surface expression of GPIX from both populations was analyzed by flow cytometry using the monoclonal anti-CD42a antibody GRP (Serotec) specific for the GPIX subunit. Fig. 2ACitation shows a clear rightward shift in the staining profile at 5 days, demonstrating an increase in the expression of GPIX on the cell surface. Surface expression was elevated as early as 2 days after stimulation; however, the rise was not as pronounced (data not shown). In contrast, the expression profile of GPIX on cells that were not exposed to PMA remained relatively unchanged over the same period (Fig. 2B)Citation .



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Fig. 2. PMA and TPO induce GPIX surface expression in MK cell lines. A and B, GPIX surface expression in Dami cells was assessed via flow cytometry with an anti-CD42a monoclonal antibody (GRP). Dami cells were growth-arrested overnight and harvested at day 0 (thin lines) and at day 5 (thick lines). The cells were then labeled with anti-CD42a (GPIX) antibody, followed by an FITC-conjugated secondary antibody and examined by flow cytometry. A, cells exposed to PMA (100 nmol/liter) produced a shift to the left after 5 days. B, nontreated cells were used as a negative control. C and D, TPO-inducible expression of GPIX on the surface of UT-7 Epo/Mpl cells was investigated using flow cytometry. Cells were subject to growth arrest overnight then incubated in basal culture medium (Iscove’s modified Dulbecco’s medium/10% FBS/1 unit/ml EPO) supplemented with 20 ng/ml TPO (C) or basal culture medium alone (D). Cells were harvested at day 0 and day 2 and processed as for A and B. Surface expression at day 0 is represented by the thin dotted line and at day 2 by the thick continuous line. GPIX surface expression remained elevated up to 7 days after TPO stimulation (data not shown). The profile of staining using the IgG1 control antibody did not change over the same time period (data not shown). Results are representative of three independent experiments.

 
TPO Modulates GPIX Surface Expression in UT-7 Epo/Mpl Cells.
The preceding findings using PMA as a differentiating agent demonstrated an increase in GPIX mRNA and protein expression on the cell surface after PMA treatment. We extended these studies to include the response of cells to a more physiologically relevant agent, TPO. For these studies, we used the factor-dependent cell line UT-7 Epo/Mpl, because Dami cells are growth factor-independent and demonstrate limited responsiveness to exogenous cytokines such as TPO (41) . To investigate whether TPO could modulate GPIX expression, we conducted flow cytometry experiments as before, this time incubating UT-7 Epo/Mpl cells with 1 unit/ml EPO alone (EPO was required in the basal culture medium to maintain cell viability) or medium containing 1 unit/ml EPO plus 20 ng/ml TPO for various periods. TPO increased GPIX surface protein expression as early as 2 days after TPO treatment with elevated expression continuing after 7 days (Fig. 2C) . In contrast, incubation in basal medium alone did not increase the level of GPIX surface expression (Fig. 2D)Citation .

PMA Induces Expression of a GPIX-Luciferase Reporter Construct, and Induced Expression Is Dependent on an Intact Ets Site.
To investigate whether PMA could induce GPIX expression at the level of transcription, we cloned a fragment of the GPIX promoter (-567 to +50) into the pGL3 Basic luciferase reporter plasmid (Promega Corporation), termed pGPIX-567 luc. This construct was transiently transfected into Dami cells, and immediately after transfection, cells were split into two populations. After overnight growth arrest, cells were subsequently treated with PMA (100 nmol/liter) or vehicle alone. Exposure of cells to PMA stimulated expression of the GPIX-driven Firefly luciferase reporter by approximately 4–5-fold in comparison with nontreated cells (Fig. 3A)Citation . To identify which elements in the promoter were responsible for the inducible response, we generated nested 5' deletions of the parent construct. Deletion of the promoter up to and including -65 relative to the transcriptional start site did not significantly alter the level of induction. However, when the region between -65 and minus]18 was deleted, PMA was no longer able to activate the GPIX promoter. Deletion of this region also significantly reduced basal levels of reporter activity. The region between -65 and -18 contains a previously identified inverted Ets site 5'-ACTTCCT-3' at position -45. Interestingly, deletion of a GATA motif located in an adjacent region between -85 and -65 also slightly attenuated but did not abolish PMA- inducible GPIX activity. To account for variations in transfection efficiency in this and subsequent experiments, all of the results were normalized to the cotransfected Renilla luciferase control plasmid under the control of the thymidine kinase promoter.



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Fig. 3. PMA-inducible GPIX reporter activity is dependent on an intact Ets motif. A, 5' nested deletions were constructed to identify the motifs responsible for PMA-inducible GPIX expression in Dami cells. B, site-directed mutagenesis was used to introduce mutations into the GATA or Ets motifs of the GPIX-luciferase reporters. Cells were transfected with 5 µg of the indicated GPIX-luciferase reporter constructs and 1 µg of pRL-TK internal control plasmid. After transfection, each sample was split into two populations and growth-arrested overnight. One population was treated with PMA (100 nmol/liter), and the other was treated with vehicle alone (DMSO). Cell extracts were harvested 2 days later, and luciferase activity was analyzed. GPIX-dependent reporter activity was normalized to Renilla luciferase activity under the control of the thymidine kinase promoter to account for variations in transfection efficiency. {square}, -PMA; {blacksquare}, +PMA. Each experiment was performed in duplicate at least three times. Results represent normalized mean ± SD of a representative experiment.

 
To further confirm the importance of the Ets site in the GPIX promoter for PMA-induced expression, site-directed mutagenesis was used to disrupt either the Ets motif or upstream GATA motif in both pGPIX-567 luc as well as the shorter construct pGPIX-85 luc, as outlined in Table 1Citation . Mutation of the Ets site in either construct abolished both basal and inducible levels of GPIX-dependent reporter activity (Fig. 3B)Citation . In contrast, mutation of the GATA site in the parent construct had no effect (Fig. 3B)Citation and attenuated basal and inducible expression directed by the shorter construct, consistent with findings from the deletion analysis (Fig. 3A)Citation . These results clearly show that although the GATA site is required for basal activity of the GPIX promoter, it is the Ets site that is absolutely required for PMA-induced expression of the GPIX gene.


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Table 1 GPIX promoter sequence mutations

 
The Ets Motif in the GPIX Promoter Is Also Required for TPO Inducible Reporter Activity.
To determine whether TPO-dependent GPIX expression was regulated at the level of transcription, UT-7 Epo/Mpl cells were transfected with GPIX-567luc, subjected to growth arrest overnight and grown in the basal growth medium containing EPO or in medium containing EPO plus TPO. Levels of GPIX-dependent reporter activity were consistently 2–3-fold greater in response to TPO as those observed in Dami cells exposed to PMA. Furthermore, TPO-induced GPIX promoter activity was abolished by deletion of the region between -65 and -18, which contained the Ets motif (Fig. 4A)Citation . Mutation of the Ets element also abrogated TPO-inducible reporter activity, whereas deletion of the region between -85 and -65 containing the GATA motif or mutation of the GATA motif decreased basal reporter activity by 2-fold but did not affect the level of induction of TPO-inducible reporter activity (Fig. 4B)Citation . Hence, TPO-induced expression of GPIX is similar to PMA, with respect to the requirement of the Ets motif in the proximal promoter for inducible expression of GPIX.



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Fig. 4. TPO-inducible GPIX-reporter activity is also dependent on an intact inverted Ets motif at -45. UT-7 EPO/Mpl cells were transfected with GPIX-reporter constructs as described in "Materials and Methods." A, UT-7 EPO/Mpl cells were transfected with nested 5'-deletions of the GPIX promoter. B, UT-7 EPO/Mpl cells were transfected with the GPIX-reporter constructs bearing mutations of either the GATA or the Ets motifs. {square}, culture in basal culture medium; {blacksquare}, culture in the presence of 20 ng/ml TPO. Results represent normalized mean ± SD and are representative of at least three independent experiments performed in duplicate.

 
Fli-1 Binds Constitutively to the Ets Motif in the GPIX Promoter.
EMSAs were used to determine the identity of the factor(s) binding to the Ets site in the GPIX promoter in response to PMA stimulation. EMSAs performed using an oligonucleotide probe corresponding to the region of the GPIX promoter containing the Ets binding site with nuclear extracts from Dami cells exposed to PMA showed a single specific nuclear protein complex (Fig. 5)Citation . The presence of this complex was independent of PMA stimulation, consistent with previous reports (39) of a lack of a PMA-inducible complex using an oligonucleotide probe from the GPIIb promoter bearing an Ets site. This constitutive complex was supershifted with an antibody against Fli-1, as reported previously (24) . In contrast, an antibody against another Ets member, PU.1, did not affect the mobility of the nuclear protein complex. This result suggests that the PMA responsive region in the proximal GPIX promoter interacts with an Ets factor antigenically related to Fli-1. As expected, oligonucleotides bearing the same mutations in the Ets element introduced into GPIX-567 luc and GPIX-85 luc were unable to compete with wild-type oligo bearing the intact Ets site (Fig. 5)Citation .



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Fig. 5. Fli-1 binds constitutively to the Ets motif in the GPIX promoter. A 32P-labeled oligonucleotide probe corresponding to the GPIX proximal promoter bearing the Ets binding site, GPIX-EBS, was incubated with nuclear extracts from starved and PMA-treated Dami cells in the presence of 100 x unlabeled competitors or antibodies as outlined in "Materials and Methods." The specific oligonucleotide-Fli-1 complexes are indicated by arrows labeled Fli-1. S, supershifted Fli-1 nuclear-protein complex; A, nonspecific nucleoprotein complex.

 
The MAPK Inhibitor PD98059 Inhibits both Basal and Inducible Expression of GPIX.
Recent studies (42, 43, 44) have demonstrated that Ets factors are regulated by MAPKs in other cell types. Because our data suggested a role for the Ets member Fli-1 in the inducible expression of GPIX, we assessed the requirement of MAPK in PMA-inducible GPIX expression. Dami cells were transfected with the parent GPIX-luciferase reporter construct before growth arrest and incubated with PMA after preincubation with the MAPK/ERK kinase inhibitor PD98059. In concurrent experiments, transfected Dami cells were preincubated with the specific p38 inhibitor SB202190 before stimulation with PMA. PD98059 inhibited both basal and inducible levels of GPIX reporter activity in a dose-dependent manner (Fig.6A)Citation . In contrast, increasing amounts of SB202190 had no significant effect on either basal or inducible levels of GPIX-dependent reporter activity. A second type of experiment was performed to confirm these observations. Dami cells were preincubated with the inhibitors as outlined above and treated with PMA (100 nmol/liter). Total RNA was harvested 12 h after PMA stimulation, and an equal amount of RNA (15 µg) from each sample was subject to Northern blotting. PMA-inducible GPIX mRNA expression was inhibited in the presence of the ERK kinase inhibitor PD98059 (30 µM) but not in the presence of the p38 inhibitor SB202190 (500 nmol/liter; Fig. 6BCitation ). These data present clear evidence for the importance of upstream activation of the ERK signaling protein of the MAPK pathway.



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Fig. 6. Basal and inducible activation of GPIX is dependent upon upstream activation of ERK. A, Dami cells were transfected with the GPIX-567 luc reporter and split into two populations. Cells were growth-arrested overnight, and both populations were preincubated with the indicated concentrations of the ERK inhibitor PD98059 or p38 inhibitor SB202190 for 30 min. One population of cells was then incubated with PMA (100 nmol/liter). Luciferase activity was determined 2 days later. {square}, -PMA; {blacksquare}, +PMA. B, after overnight growth arrest, Dami cells were incubated with PD98059 (30 µM) or SB202190 (500 nmol/liter) for 30 min before PMA (100 nmol/liter) treatment. Total RNA was harvested at 12 h, and GPIX mRNA expression was analyzed by Northern blot. Membranes were rehybridized with [32P] GAPDH cDNA to demonstrate the amount of RNA loaded. C, UT-7 EPO/Mpl cells were transfected with the GPIX reporter gene construct GPIX-567 luc. After overnight growth arrest, cells were treated with the above inhibitors at the indicated concentrations for 4 h before TPO exposure. Luciferase activity was determined 2 days later as outlined in "Materials and Methods." {square}, culture in basal culture medium; {blacksquare}, culture in the presence of 20 ng/ml TPO. All of the transfection results represent normalized mean ± SD and are representative of three independent experiments performed in duplicate.

 
TPO-inducible GPIX Expression Requires Upstream Activation of ERK.
Binding of TPO to its receptor c-Mpl activates a number of different intracellular signaling pathways through the activation of nonreceptor kinases that include Jak-Stat, PKC, and MAPKs (29, 30, 31, 32, 33, 34) . UT-7 Epo/Mpl cells transfected with pGPIX-567luc were pretreated with different signaling inhibitors before TPO exposure to identify which particular pathway was required for TPO-inducible GPIX reporter activity. As was the case for PMA in Dami cells, preincubation of UT-7 Epo/Mpl with increasing amounts of PD98059 decreased both basal and TPO-inducible GPIX-dependent reporter activity in a dose-dependent manner, although ERK inhibition did not abrogate GPIX induction completely. In contrast, neither SB202190 nor the PKC inhibitor GF109203X had any inhibitory effect on GPIX-dependent reporter activity whatsoever (Fig. 6C)Citation . Taken together, these data demonstrate the importance of ERK signaling and the 5'-ACTTCCT-3' element for both basal GPIX activity and inducible expression of GPIX.

Fli-1-dependent Activation of GPIX Is Independent of de Novo Protein Synthesis or Phosphorylation.
Additional studies were conducted to determine the mechanisms involved in Erk-dependent GPIX expression. First, Northern blotting was performed to assess the levels of Fli-1 mRNA in response to PMA treatment in the presence of the MAPK and p38 inhibitors. PMA-treatment of Dami cells had no effect on the level of Fli-1 mRNA in the absence of any inhibitors. Additionally, pretreatment of Dami cells with either PD98059 or SB202190 did not effect Fli-1 mRNA (Fig. 7)Citation or protein levels (data not shown). Considering PMA treatment did not result in inducible expression of Fli-1 or in the formation of an inducible binding complex at the Ets motif in the GPIX promoter (Fig. 5)Citation , we reasoned that other events such as phosphorylation of Fli-1 may be involved. Western blotting using nuclear extracts from PMA-treated Dami cells failed to identify the formation of a higher molecular-weight species indicative of phosphorylated immunoreactive Fli-1, and there was no discernible change in Fli-1 electrophoretic mobility after treatment with calf intestinal phosphatase (data not shown). Moreover, to rule out any effect upon Fli-1 DNA-binding activity by the MAPK inhibitors, EMSA was performed using extracts from cells pretreated with either PD98059 or SB202190. The specific nuclear-protein complex identified as Fli-1 remained unchanged in cells pretreated with either the MAPK (Fig. 8)Citation or p38 inhibitors (data not shown). These results suggest that direct phosphorylation of Fli-1 does not play a role in activation of GPIX, yet binding and functional analyses demonstrate that its occupancy at the promoter is critical for both basal and inducible GPIX expression.



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Fig. 7. Fli-1 mRNA is unaffected by PMA treatment or blockade by PD98059. After overnight growth arrest, Dami cells were incubated with PD98059 (30 µ M) or SB202190 (500 nmol/liter) for 30 min before PMA (100 nmol/liter) treatment. Total RNA was harvested at 12 h, and Fli-1 mRNA expression was analyzed by Northern blot. Membranes were rehybridized with [32P]GAPDH cDNA to demonstrate the amount of RNA loaded.

 


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Fig. 8. MAPK inhibition by PD98059 does not affect Fli-1 binding activity. EMSA was performed using nuclear extracts from Dami cells pretreated with PD98059 or SB202190 (data not shown) and subjected to PMA stimulation in the presence of anti-Fli-1 antibody as outlined in "Materials and Methods." The specific oligonucleotide-Fli-1 complexes are indicated by arrows labeled Fli-1. S, supershifted Fli-1 nuclear-protein complex; A, nonspecific nucleoprotein complex.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Expression of GPIX is an important step during MK differentiation. The GPIX subunit is required for both adequate and functional expression of the entire GPIb-IX complex on the MK/platelet surface (15) . This complex contains the vWf binding site for initial adhesion of platelets to the vessel wall during bleeding (14) . Recent evidence (21) also suggests that expression of the GPIb-IX complex on the cell surface results in activation of intracellular signaling events that may regulate MK growth. Hence, we were interested to study the factors and signaling events that regulate GPIX expression during MK differentiation.

The ability of PMA and TPO to induce differentiation of megakaryocytic cell lines is well established. Along with changes in cell morphology, increase in nuclear content, and cell size, PMA and TPO are able to induce the expression of MK markers such as GPIIb, GPIIIa, and GPIb{alpha} (36 , 37 , 40) . However, to our knowledge the expression profile of GPIX in response to either PMA or TPO has not been documented. Therefore, we initially set out to demonstrate the ability of PMA and TPO to modulate GPIX expression at mRNA and protein level. PMA was able to induce the expression of GPIX mRNA as early as 12 h after stimulation, with the level of induction becoming more marked up to 48 h. Induced expression of GPIX mRNA also required new protein synthesis, because the response in the presence of cycloheximide was totally abolished. Induction of GPIX expression at mRNA level was also reflected by an increase in surface protein expression as analyzed by flow cytometry at both 2 and 5 days. Results using the more physiologically relevant cytokine TPO in UT-7 Epo/Mpl cells were similar to that of PMA in Dami cells, with increased surface expression of GPIX evident after 2 days and continuing at 7 days. The increase in GPIX mRNA and surface expression follows similar time courses to those observed for other MK markers after PMA and TPO treatment, such as GPIIb and GPIb{alpha}. It is likely that expression of genes of the GPIb-IX complex are regulated by similar mechanisms, because of the requirement of coordinated expression of subunits of the GPIb-IX complex on the MK/platelet surface, despite the fact that the genes are located on different chromosomes (15) .

The presence of Ets and GATA motifs are by far the most distinguishing features of promoter regions of MK-specific genes (6) . The same is true for the GPIX promoter, the constitutive expression of which has been characterized by Bastian et al. previously (11) in HEL cells. However, our interest in the GPIX promoter was to identify motifs important for induced expression using the differentiating agents PMA and TPO. Although deletion of upstream elements up to position -65 relative to the GPIX transcription start site affected basal levels of reporter activity, they had no significant effect on the ability of either PMA or TPO to induce expression of the GPIX-reporter construct. This region included the GATA site identified by Bastian et al. previously (11) as being important for expression under basal conditions.

Consistent with previous findings (11) , deletion or mutation of the GATA site in the GPIX promoter did reduce the levels of basal reporter activity by approximately 2-fold; however, deletion or mutation of the GATA site did not have any significant effect on the fold increase of GPIX-reporter activity by PMA or TPO. In contrast, deletion or mutation of the inverted Ets motif 5'-ACTTCCT-3' at position -45 totally abolished levels of both basal and inducible GPIX-dependent reporter activity. TPO-induced expression of another MK-specific gene, GPIIb, has also been demonstrated to involve an Ets site at position -514 (25) . Furthermore, in the same study it was demonstrated that mutation of an adjacent GATA site did not effect TPO-inducible GPIIb activity. Studies using GATA-1 null cells and others involving overexpression of GATA-1 in cell line models clearly demonstrate the importance of GATA-1 in both MK and erythroid development (45, 46, 47) . The fact that deletion or mutation of the GATA site in the GPIX promoter did not significantly effect the fold induction by PMA or TPO was somewhat surprising, although the expression of GATA-1 mRNA has been reported to be down-regulated in response to stimulation by phorbol esters in the MK cell line HEL (48) . Our observations are also consistent with recent work by Zutter et al. (49) who observed a lack of enhancer function of the GATA site in the 5' flanking region of the {alpha}2 integrin gene. Era et al. (50) recently suggested that some Ets members, rather than members of the GATA family, may function to restrict expression of MK-specific genes to the megakaryocytic lineage. The absolute requirement of an intact Ets binding site for PMA and TPO-inducible expression of the GPIX gene provides further support for this model.

The Ets family member Fli-1 has been shown previously (24) to bind specifically to the Ets motif in the GPIX promoter and is able to transactivate the GPIX, GPIIb, and GPIb{alpha} promoters in 293T cells. Overexpression of Fli-1/Erg B in K562 cells induces morphological changes characteristic of MK differentiation (26) . Furthermore, Fli-1 has been identified in MK cell lines, primary MKs, and platelets (24 , 28) . This evidence provides firm support for Fli-1 as a potential candidate for mediating PMA and TPO-inducible expression of GPIX. Indeed, our gel shift and mutational analysis confirmed that Fli-1 bound to the 5'-ACTTCCT-3' element in the GPIX promoter critical to PMA and TPO-inducible gene expression.

The signaling pathways involved in PMA and TPO-inducible expression of GPIX have not been determined. Ets family members are known targets of the MAPK signaling pathway in other cell types (42, 43, 44) , and the MAPK pathway has been implicated in MK differentiation by a number of investigators (49) . Moreover, binding of TPO with its receptor c-Mpl activates a number of different signaling pathways, which include the ras/raf/MAPKK1/MAPKK2/ERK cascade (33) . Our studies demonstrated the ERK inhibitor PD98059 was able to decrease both PMA- and TPO-induced expression of a GPIX-luciferase reporter construct and endogenous GPIX expression. In contrast, inhibition of p38/Jnk or PKC had no effect on inducible GPIX activity.

The mechanism of ERK-dependent GPIX expression and how it relates to the necessity of Fli-1 at the GPIX promoter is unclear at the present time. We determined that Fli-1 occupancy of the 5'-ACTCCT-3' element in the proximal GPIX promoter is crucial for basal and PMA/TPO-inducible expression but were unable to observe an inducible nucleoprotein complex after exposure to PMA or TPO (data not shown), despite using multiple binding conditions. Fli-1 expression was also unchanged in response to PMA treatment in Dami cells, and blockade of ERK by PD98059 had no effect on Fli-1 expression or DNA-binding activity. We hypothesized that PMA or TPO may act through phosphorylation of Fli-1. However, Western blotting using nuclear extracts from PMA-treated Dami cells failed to identify the formation of a higher molecular species indicative of phosphorylated immunoreactive Fli-1 (data not shown). These results suggest that direct phosphorylation of Fli-1 does not play a role in activation of GPIX, yet our binding and functional analyses demonstrate that its presence at the promoter is critical for GPIX expression.

Taken together, the above findings suggest that Fli-1 may undergo other post-translational modifications at the GPIX promoter or that it plays a key role in combinatorial interactions involving other as yet unidentified transcription factors. Fli-1 has been found previously (51) to participate in multiprotein complexes at functionally important recognition elements in other promoters. Moreover, Fli-1 has been shown to physically interact with AP-1 family members in other cell types (52) that themselves have been demonstrated to be targets of ERK-dependent signaling (53) . Therefore, we propose an alternative mechanism wherein Fli-1, bound to the promoter, recruits other accessory protein(s) upon PMA or TPO stimulation, which are themselves activated by ERK. Additional studies should clarify potential partners of Fli-1 that may be involved in ERK-dependent expression of the GPIX gene during MK differentiation.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cell Culture.
Dami cells were obtained from the American Type Culture Collection and routinely cultured in RPMI 1640 supplemented with 2.0 g/liter sodium bicarbonate and 10% FBS (Life Technologies, Inc.) in a humidified 37°C 5% CO2 incubator. Cells were passaged every 3–4 days. Growth factor-dependent UT-7 EPO cells stably transfected with the TPO receptor c-Mpl (UT-7 Epo/Mpl) were kind gifts from Dr. Norio Komatsu (Jichii Medical School, Tochigi, Japan) and were cultured as described previously (54) in Iscove’s modified Dulbecco’s medium supplemented with 3.024 g/liter sodium bicarbonate, 10% FBS, 1 unit/ml EPO, and 1 mg/ml geneticin.

RNA Isolation and Northern Blotting.
Exponentially growing Dami cells were seeded at a density of 5.0 x 105/ml and growth-arrested overnight in the appropriate media containing 0.5% FBS. Cells were then exposed to PMA and total RNA harvested using TRIzol RNA isolation reagent (Life Technologies, Inc.) according to the manufacturer’s instructions at the indicated time points. Total RNA (15 µg) was subject to electrophoresis on a 1% formaldehyde-agarose gel and transferred to Hybond N+ nylon membrane (Amersham). Membranes were prehybridized for 5 h at 42°C. Hybridization was performed overnight at 42°C with the described cDNA probes that had been radio-labeled with 20 µCi of [{alpha}32P]dCTP (GeneWorks) using nick translation and separated from unincorporated label using Sephadex G-50 quick spin columns (Boehringer Mannheim). Membranes were washed in 2 x SSC/0.5% SDS and 0.2 x SSC/0.5% SDS at 42°C and then subjected to autoradiography at -80°C.

Generation of Oligonucleotide Probes for Northern Blotting.
The probe corresponding to full-length GPIX cDNA was generated by digesting the pBluescriptSK+-GPIX plasmid (American Type Culture Collection) with EcoRI. The resulting 604-bp fragment was gel purified before use. The Fli-1 probe corresponded to a 700-bp cDNA fragment encompassing the Ets domain and 3' COOH-terminal activation of Fli-1. The GAPDH cDNA probe corresponded to a 316-bp fragment of the human GAPDH-coding sequence derived from exons 5–8 (Ambion, Austin, Texas) that had been inserted into the EcoRI/PstI sites of the pUC19 plasmid. The pUC19-GAPDH plasmid was digested with EcoRI and PstI, and the GAPDH fragment gel was purified and labeled as described above.

Flow Cytometry.
Flow cytometry was performed on a FACStar Plus cytometer (Becton Dickinson) as described previously (55) . Cells in suspension were removed and pelleted by centrifugation at 180 x g. Any adherent cells were detached from culture dishes with 0.5 mmol/liter EDTA in PBS and combined with the corresponding suspension cells. Cells were then washed three times in PBS/1% BSA. The cells (2.0 x 105/reaction) were incubated with anti-CD42a monoclonal antibody GRP (IgG1; Serotec) or an irrelevant mouse IgG1 control antibody (against keyhole limpet hemocyanin; Becton Dickinson) for 10 min at room temperature, washed three times in 1% BSA/PBS, and then incubated at room temperature with the relevant FITC-conjugated secondary antibody (goat antimouse IgG at a 1:200 dilution). Cells were washed three times and were kept in the dark until analysis.

Construction of GPIX Luciferase Reporter Constructs.
The promoter region of GPIX -567 to +50 was generated using the PCR with the sense oligonucleotide primer 5'-ACGCGTCACTTGGTAGAGGCTCTG-3' and the antisense oligonucleotide primer 5'-AGATCTGCTCCTGTCTGGCCTGAA-3' from human genomic DNA (Boehringer Mannheim). The resulting 630-bp fragment was subcloned into the pGL3 basic luciferase reporter plasmid (Promega Corporation) using the MluI/BglII restriction sites incorporated into the 5' ends of the described oligonucleotide primers to create pGPIX-567luc. DNA sequencing was used to confirm the integrity of the GPIX promoter sequence. Nested 5' deletions of the parent pGPIX-567luc plasmid for constructs up to and including pGPIX-155 luc were generated using exonuclease III. The remaining three deletions, pGPIX-85 luc, pGPIX-65 luc, and pGPIX-18 luc, were generated by PCR using pGPIX-567 luc as the template and the following respective sense oligonucleotide primers: 5'-AATACGCGTCAAGGCTGCACTGGG-3', 5'-GGACGCGTAAGCCAGGCTATTTTCATCAC-3', and 5'-CGACGCTCCACCTCAGCTGTATCC-3', with the antisense primer 5'-CACTGCATACGACGATTCTGT-3'. The resulting PCR products were then digested with MluI/BglII and subcloned into the pGL3 basic plasmid, which had been digested with the same enzymes. Mutations of the GATA site at position -67 and the Ets site at position -45 were made using the Quik-change site-directed mutagenesis kit (Stratagene) with the following sense and antisense oligonucleotide primers respectively: 5'-CTGCACTGGGGGAATTCGCCAGGC-3', 5'-GCCTGGCGAATTCCCCCAGTGCAG-3', 5'-ATTTTCATCACATACCTGCGCCCGCTCCCG-3', and 5'-CGGGAGCGGGCGCAGGTATGTGATGAAAAT-3'. Oligonucleotides were synthesized by Life Technologies, Inc. The integrity and location of deletions and mutations of the promoter sequence were confirmed by automated DNA sequencing.

Transient Transfection.
Transient transfection of both Dami and UT-7 cells was carried out using N-[1-(2,3-dioleoyloxyl)propyl]-N,N, N-trimethylammoniummethyl (DOTAP) sulfate liposomal transfection reagent (Boehringer Mannheim) as described previously (11) . Dami cells were transfected with 5 µg of the indicated reporter plasmid and 1 µg of pRL-TK internal control plasmid (Promega Corporation) in 100-mm culture dishes. After transfection, cells were split into two populations and seeded into 6-well plates. Cells were subject to growth arrest overnight, followed by stimulation with 100 nmol/liter PMA dissolved in DMSO or DMSO alone as control. UT-7 cells were transfected with 2.5 µg of the indicated reporter plasmid and 50 ng of pRL-TK internal control plasmid in 6-well plates. After transfection and growth arrest, UT-7 cells were treated with either 1 unit/ml EPO or 1 unit/ml EPO + 20 ng/ml TPO. Cell extracts were harvested 48 h after stimulation, washed once in PBS, and resuspended in 200 µl of 1 x passive lysis buffer supplied with the Dual Luciferase Assay Reporter System (Promega Corporation). Cells were subject to three cycles of freeze-thaw lysis. Lysate (20 µl) was used for the luciferase assay. Luciferase assays were performed using the dual luciferase assay reporter system according to the manufacturer’s instructions in a Turner Designs TD20/20 luminometer. Each assay was performed in duplicate at least three times.

EMSAs.
Oligonucleotides bearing the GPIX Ets binding site, 5'-ATTTTCATCACTTCCTTCCGCCCGCTCCCG-3', and its complement were annealed and end-labeled using polynucleotide kinase and [{gamma}-32P]ATP, followed by purification on chromaspin-10 spin columns according to the manufacturer’s instructions (Clontech Laboratories, Paolo Alto, CA). Nuclear extracts were harvested from 1.0 x 106 cells using the method of Schrieber et al. (56) . Binding reactions were performed using 5–10 µg of Dami nuclear extracts using conditions as described previously (57) in a total volume of 20 µl on ice for 20 min. In samples containing Fli-1 or PU.1 antibodies (BD PharMingen, San Diego, CA) or excess unlabeled competitor, 2 µl of antibody or 1 µl of competitor was added 15 min before the addition of the probe. After binding, the reaction was subject to PAGE on 5% polyacrylamide gels in 0.5 x Tris-borate EDTA at 4oC. Gels were dried and exposed to Kodak (Rochester, NY) Hyperfilm MP at -80oC with an intensifying screen.


    Acknowledgments
 
We thank Leonie Gaudry (Prince of Wales Hospital, Sydney, Australia) for assistance in conducting the flow cytometry experiments and Dr. Norio Komatsu (Jichii Medical School, Tochigi, Japan) for kindly providing the UT-7-Epo/Mpl cell line.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by a grant from the National Health and Medical Research Council and New South Wales Department of Health. Back

2 These two authors contributed equally to this work. Back

3 To whom requests for reprints should be addressed, at Department of Medicine, Clinical Sciences Building, St. George Hospital, Kogarah NSW, 2217 Australia. Phone: 61-2-9350-2010; Fax: 61-2-9350-3998; E-mail: b.h.chong{at}unsw.edu.au. Back

4 The abbreviations used are: MK, megakaryocyte; GP, glycoprotein; TPO, thrombopoietin; vWf, von Willebrand factor; MAPK, mitogen-activated protein kinase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; ERK, extracellular signal-regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EPO, erythropoietin; EMSA, electrophoretic mobility shift assay; FBS, fetal bovine serum. Back

Received for publication 12/11/00. Revision received 5/24/01. Accepted for publication 6/15/01.


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