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Autocrine signaling of platelet-derived growth factor regulates disabled-2 expression during megakaryocytic differentiation of K562 cells
FEBS 29792
FEBS Letters 579 (2005) 4395–4401
Autocrine signaling of platelet-derived growth factor regulates disabled-2 expression during megakaryocytic differentiation of K562 cells Ching-Ping Tsenga,b,*, Pam Changa, Chien-Ling Huanga, Ju-Chien Chengc, Shy-Shin Changd b
a Graduate Institute of Basic Medical Sciences, Chang Gung University, Tao-Yuan 333, Taiwan, ROC Graduate Institute of Medical Biotechnology, Chang Gung University, 259 Wei-Hwa 1st Road, Kwei-Shan, Tao-Yuan 333, Taiwan, ROC c School of Medical Laboratory Sciences and Biotechnology, China Medical University, Taichung 404, Taiwan, ROC d Department of Emergency Medicine, Chang Gung Memorial Hospital, Tao-Yuan 333, Taiwan, ROC
Received 17 January 2005; revised 25 June 2005; accepted 27 June 2005 Available online 19 July 2005 Edited by Veli-Pekka Lehto
Abstract Platelet-derived growth factor (PDGF) is involved in megakaryocytopoiesis and is secreted into the culture medium during megakaryocytic differentiation of human leukemic cells. We investigate whether PDGF plays a role in the regulation of the adapter protein Disabled-2 (DAB2) that expresses abundantly in platelets and megakaryocytes. Western blot analysis revealed that conditioned medium from 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated, megakaryocytic differentiating K562 cells upregulated DAB2 expression. DAB2 induction and megakaryocytic differentiation was abrogated when cells were co-treated with the PDGF receptor inhibitor STI571 or when the conditioned medium was derived from TPA-plus STI571treated cells. Although the level of PDGF mRNA was not altered by STI571, an approximate 44% decrease in PDGF in the conditioned medium was observed. Consistent with these findings, interfering PDGF signaling by PDGF neutralization antibody or dominant negative PDGF receptors attenuated DAB2 expression. Accordingly, transfection of an expression plasmid encoding secreted PDGF upregulated DAB2. This study shows for the first time that PDGF autocrine signaling regulates DAB2 expression during megakaryocytic differentiation. 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Disabled-2; Megakaryocytic differentiation; Platelet-derived growth factor; STI571
1. Introduction Platelet-derived growth factor (PDGF) constitutes a family of peptide growth factors that includes at least four dimeric forms, PDGF-AA, -AB, -BB, and -CC [1]. PDGF dimer is linked together by disulfide bonds and activates two specific receptors, the PDGF a-receptor (PDGFRa) and the PDGF b-receptor (PDGFRb). In blood, PDGF expresses abundantly in megakaryocyte and platelet a-granule. Targeting disruption of PDGF-B or PDGFRb results in various hematopoietic defects, including microvessel leakage, hemorrhage, microaneurysm formation, anemia, and thrombocytopenia [2,3]. Apart
from its pivotal role in thrombosis and hemostasis, PDGF is a positive regulator for megakaryocytopoiesis. During megakaryocytic differentiation of human leukemic cells, PDGF-A and PDGF-B are induced through gene activation and are secreted into the culture medium together with other autocrine regulatory factors [4,5]. The conditioned medium retains the ability to induce megakaryocytic differentiation [6], with PDGF signaling mediates the expression of megakaryocyte-associated transcription factors NF-E2, GATA-1, and c-FOS [7]. PDGF is thus considered as one of the megakaryocytic lineage determination factors present in the conditioned medium. Disabled-2 (DAB2) is an adapter protein that is implicated in growth factor signaling [8,9], endocytosis [10–12], and cell adhesive function [13,14]. During in vitro megakaryocytic differentiation of human leukemic cells and human umbilical cord blood CD34+ hematopoietic stem cells, DAB2 is upregulated and acts as a negative regulator in integrin a IIbb3-mediated fibrinogen adhesion and cell signaling [13,15,16]. DAB2 is also induced during visceral endoderm and gastrointestinal development and retinoic acid-induced F9 cell differentiation [17–19]. Of these differentiation models, DAB2 is mainly regulated at the transcriptional level. However, our previous study [16] in comparing the extent of DAB2 mRNA and protein expression during megakaryocytic differentiation of K562 cells implicates the presence of an additional level of regulation in the control of DAB2 gene expression that has not yet been characterized. With the tight association between DAB2 expression and megakaryocytic differentiation and the presence of megakaryocytic lineage determination factors in the conditioned medium, we address whether the autocrine regulatory factors plays a role in DAB2 expression. In this study, we report that the conditioned medium from megakaryocytic differentiating cells retains the ability to induce DAB2 and implicates a link between DAB2 expression and autocrine signaling. Through molecular targeting approaches using PDGF receptor inhibitor STI571 [20], dominant negative PDGF receptors and expression of recombinant PDGF, we further demonstrate that the autocrine signaling of PDGF plays a crucial role in the regulation of DAB2 expression mainly through a non-transcriptional control mechanism. 2. Materials and methods
*
Corresponding author. Fax: +886 03 2118355. E-mail address: [email protected] (C.-P. Tseng). Abbreviations: DAB2, Disabled-2; PDGF, platelet-derived growth factor; TPA, 12-O-tetradecanoylphorbol-13-acetate
2.1. Materials The anti-b-actin antibody and TPA were purchased from Sigma Chemical Co. (St. Louis, MO). The anti-DAB2 antibody p96 was purchased from BD Transduction Laboratories (San Jose, CA). The
0014-5793/$30.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.06.080
4396 anti-b3, CD61, PDGFRa, and PDGFRb antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-p42/p44 MAPK and phospho-p42/p44 MAPK antibodies were purchased from New England Biolab (Beverly, MA). Quantikine human PDGF-BB immunoassay and the anti-PDGF neutralization antibody were from R&D Systems Inc. (Minneapolis, MN). The STI571 (Gleevec) is the product of Novartis Pharma AG (Basel, Switzerland). The 2· SYBR Green I PCR master mix was purchased from Applied Biosystems (Foster City, CA). The kinase-inactive K627A PDGFRa and K634A PDGFRb expression plasmids [21] were a kind gift from Dr. CarlHenrik Heldin (Ludwig Institute for Cancer Research, Uppsala, Sweden). The truncated PDGFRa expression plasmid [22] was a kind gift from Dr. Andrius Kazlauskas (The Schepens Eye Research Institute, Harvard Medical School, Boston, MA). 2.2. Plasmid construction To generate pcDNA3-D-PDGFRa, a 2.0-kb NotI–ApaI DNA fragment corresponding to the truncated PDGFRa in pLXSN vector [22] was subcloned into the pcDNA3 expression vector. To construct pSecTag2A-PDGFB, PDGF-B cDNA was obtained from K562 cells by RT-PCR using the primer set PDGFB-F (5 0 -AAGCTTTCTGGGTTCCCTGACCAT-3 0 ) and PDGFB-R (5 0 -GGATCCTCGAGGCTCCAAGGGT-3 0 ). The full-length PDGF-B cDNA was then subcloned into the BamHI–HindIII sites of the pSecTag2A expression vector (Invitrogen). 2.3. Cell culture, conditioned medium collection, transient transfection, and Western blot analysis K562 and OEC-M1 cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and antibiotics. The conditioned medium was obtained from the supernatant of K562 cells with the indicated treatment after centrifugation at 1500 rpm for 5 min. Transient transfection, preparation of total cell lysates, and Western blot analysis were performed as described previously [13]. 2.4. Analysis of CD61 expression by flow cytometry K562 cells were collected and washed by FABS buffer (1· PBS, 1% FBS, and 0.1% NaN3). The CD61 mouse monoclonal antibody (2 lg/ ml) was added to the cell suspension followed by incubation with fluorescein isothiocyanate-conjugated goat anti-mouse antibody (0.4 lg/ml). After several washes with the FABS buffer, K562 cells were subjected to flow cytometric analysis with FACSCAN and the data were analyzed using the CellQuest software (Becton Dickinson, San Jose, CA).
C.-P. Tseng et al. / FEBS Letters 579 (2005) 4395–4401 assay diluent buffer. The mixture was then added into a 96-wells plate that was pre-coated with PDGF-Rb/Fc chimera. Following incubation with the substrate solution, the stop solution was added and the OD450 was determined using a microplate reader. 2.8. Neutralization of PDGF activity The PDGF activity in the conditioned medium was neutralized by incubating the medium with anti-PDGF neutralization antibody or control IgG antibody for 30 min. The medium was then used to culture exponential growing K562 cells to determine the effect of PDGF on DAB2. 2.9. Establishment of dominant negative PDGFRa stable cell lines K562 cells were transfected with 6 lg of the kinase-inactive K627A PDGFRa or the truncated PDGFRa (pcDNA3-D-PDGFRa) expression plasmids. After neomycin selection (400 lg/ml) for 1 month, the stable cell lines expressing kinase-inactive or truncated PDGFRa were identified by Western blot analysis using anti-PDGFRa antibody.
3. Results 3.1. TPA-conditioned medium induces DAB2 expression and megakaryocytic differentiation Previous studies have shown that the conditioned medium from TPA-treated K562 cells mimics the effect of TPA and induces integrin b3 expression and morphological changes associated with megakaryocytic differentiation. To determine whether the conditioned medium modulates DAB2 expression, 1–3 days TPA-conditioned medium was collected and applied to the exponential growing K562 cells. Western blot analysis revealed that DAB2 was induced with maximum induction by the medium that has been ‘‘conditioned’’ for 72 h (Fig. 1A). The cells also demonstrated a typical differentiation phenotype with the characteristics of cell enlargement, decrease in N/C ratio, and nucleus with multiple lobes (data not shown). As a control, the conditioned medium derived from the cells treated with solvent control ethanol had no effect on DAB2 expression and megakaryocytic differentiation.
2.5. Real-time quantitative RT-PCR The one-step real-time quantitative RT-PCR was performed using the GenAmp 5700 Sequence Detection System (Applied Biosystems, Foster City, CA). Briefly, 300 ng of total RNA was added into a reaction mixture containing 25 ll of 2· SYBR Green I PCR master mix, 1.25 ll of 40· reverse transcriptase mixture, 1 ll of forward primer Dab2-F (100 nM, 5 0 -AGTGAGGCCCTAAGGATTCTAGATGACCAAACTA-3 0 ), and 1 ll of reverse primer Dab2-R (100 nM, 5 0 -CAGGATATCTTTGCTTTCTGTTGGACTATTTAGGTC-3 0 ) in a final volume of 50 ll. The condition for RT-PCR was 1 cycle of 48 C for 30 min, 1 cycle of 95 C for 10 min, and 40 cycles of 95 C for 15 s and 60 C for 1 min. The cycle threshold value (CT) was used to assess the quantity of target gene. The relative fold difference for the expression of DAB2 mRNA was normalized by the expression of b-actin and was determined using the value of 2DDCT. 2.6. Luciferase activity assay The K562 cells (4 · 105) were transiently transfected with 200 ng of DAB2 promoter firefly luciferase reporter construct phDab2-luc [16] and 2 ng of Renilla luciferase expression plasmid pRL-TK. After indicated treatment, the cell lysates were subjected to luciferase activity assay according to the manufacturerÕs instructions. The firefly luciferase activity was normalized by the Renilla luciferase activity in each individual sample. 2.7. PDGF quantification The human PDGF-BB immunoassay (R&D Systems Inc.) was used to quantify the amount of PDGF in the conditioned medium. Briefly, 100 ll of PDGF standard or sample medium was mixed with 100 ll of
Fig. 1. TPA-conditioned medium induces DAB2 expression. (A,B) K562 cells (5 · 104/ml) were treated with TPA (T, 10 ng/ml) or solvent control ethanol (E) for the indicated time (panel A). Alternatively, K562 cells were incubated with E or T for the indicated time and the medium was replaced by fresh RPMI 1640 medium without TPA for a total of 72 h (panel B). Conditioned medium was then collected and applied to exponential growing K562 cells (1.5 · 104/ml) for 48 h. Total cell lysates were collected and subjected to Western blot analysis with anti-p96 (DAB2) antibody. The expression of b-actin was used as a control for equal protein loading. Essentially similar results were obtained in 3 independent experiments.
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To eliminate the concern that DAB2 induction is due to TPA carryover, we modified the way for conditioned medium preparation in the following experiment. At 6, 12, and 24 h after TPA treatment, the medium was removed and replaced by fresh medium without TPA. The conditioned medium was then collected at 72 h after initial TPA treatment and applied to exponential growing K562 cells. Western blot analysis revealed that DAB2 was induced to the level comparable to the medium that has been conditioned for 72 h (e.g., treatment for 72 h without removal of TPA) (Fig. 1B). These results rule out TPA carryover as the cause of DAB2 induction and implicate the presence of soluble DAB2 regulator(s) in the TPA-conditioned medium.
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3.2. STI571 inhibits TPA- and conditioned medium-induced DAB2 expression and megakaryocytic differentiation PDGF is involved in megakaryocytopoiesis and is secreted into the culture medium during megakaryocytic differentiation of human leukemic cells. We co-treated K562 cells with TPA and PDGF signaling inhibitor STI571 to investigate whether PDGF plays a role in the regulation of DAB2. At the concentrations between 0.01 and 1 lM, STI571 inhibited DAB2 expression in a dose-dependent manner. More than 80% of DAB2 expression was inhibited by 1 lM of STI571 in both K562 cells (Fig. 2A) and megakaryocytic cell line HEL (data not shown). The conditioned medium-induced DAB2 expression was also suppressed by STI571 (Fig. 2B). Because the
Fig. 2. STI571 inhibits DAB2 expression and megakaryocytic differentiation. (A) STI571 inhibits DAB2 and integrin b3 expression in a dosedependent manner. K562 cells (5 · 104/ml) were pretreated with the indicated concentrations of STI571 for 15 min followed by 48 h treatment with TPA (T, 10 ng/ml) or solvent control ethanol (E, 0.01%). Total cell lysates were subjected to Western blot analysis with anti-p96 (DAB2) and antiintegrin b3 antibodies. The expression of b-actin was used as a control for equal protein loading. B. STI571 inhibits conditioned medium-induced DAB2 expression. Either general K562 cell culture medium or TPA-conditioned medium was collected and applied to exponential growing K562 cells for 72 h, in the presence of 100 nM STI571 (S), 10 ng/ml TPA (T), or solvent vehicle control (E). Total cell lysates were collected and subjected to Western blot analysis with anti-p96 (DAB2) and anti-b-actin antibodies. C. Effect of STI571 on TPA-induced DAB2 mRNA expression. The K562 cells (5 · 104/ml) were treated with vehicle ethanol, TPA, or TPA + STI571 (100 nM) for 48 h. The expression level of DAB2 mRNA was determined by real-time RT-PCR using 300 ng total RNA and was normalized by the levels of b-actin mRNA. D. STI571 does not inhibit TPA-induced DAB2 promoter activity. The K562 cells (4 · 105) were transfected with phDAB2-luc (200 ng) and the Renilla luciferase expression plasmid pRL-TK (2 ng) by the DMRIE-C transfection reagent. After treatment with ethanol (0.01%), TPA (10 ng/ml), and TPA + STI571 (100 nM) for 24 h, the cell lysates were collected for luciferase activity assay using the dual-luciferase reporter assay system. The DAB2 promoter activity was normalized by the Renilla luciferase activity and the normalized activity for the ethanol-treated control group was arbitrary set as 1. The data represent the mean ± S.E. of three assays in a representative experiment. Similar results were obtained in three independent experiments. E and F. Effects of STI571 on TPA-induced CD61 expression and cell morphology. K562 cells were treated with ethanol, TPA, and TPA plus STI571 for 48 h. Flow cytometric analysis was performed to detect the megakaryocytic differentiation surface marker CD61 (panel E). Aliquots of the cell suspension were cytospin, stained with Liu stain reagents, and observed by light microscopy. A typical field was shown for K562 cells treated with ethanol (control), TPA, and TPA + STI571, respectively (panel F).
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survival rate for the cells treated with STI571 was more than 95%, it ruled out non-specific cellular toxicity as the cause for the decrease in DAB2. Whether STI571 affects DAB2 transcription was determined by both real-time RT-PCR and promoter activity analysis. TPA induced a 12-fold increase in DAB2 mRNA. In the presence of STI571, a moderate 30% decrease in TPA-induced DAB2 mRNA was observed (Fig. 2C). To confirm this finding, we transfected phDab2-luc, a TPA-responsive DAB2 promoter luciferase reporter construct, into K562 cells followed by the indicated treatment (Fig. 2D). Consistent with the results of real-time RT-PCR, TPA induced approximate 15fold increase in phDab2-luc activity and the induction was not significantly altered by STI571. With the distinct extent of inhibition in DAB2 mRNA and protein, these data implicate that post-transcriptional control may be involved in STI571-mediated inhibition of DAB2 expression. In addition to modulate DAB2, integrin b3 expression was also diminished by STI571 (Fig. 2A). To elucidate the effect of STI571 on megakaryocytic differentiation, flow cytometric analysis was performed to detect the expression of megakaryocytic differentiation surface marker CD61. Our results revealed that CD61 was reduced to 23% for TPA/STI571-treated cells compared to 40% for TPA-treated cells (Fig. 2E). Accordingly, the typical phenotypes of megakaryocytic differentiation were less prominent in the TPA/STI571-treated cells (Fig. 2F). These results indicate that sub-apoptotic concentration of STI571 also impedes megakaryocytic differentiation of K562 cells. 3.3. STI571 reduces PDGF secretion into the culture medium To investigate whether STI571 mediates its effect by modulating autocrine signaling, the conditioned medium from TPA/STI571-treated cells was collected at 72 h after treatment and applied to the exponential growing K562 cells. As shown in Fig. 3A, this type of conditioned medium did not induce DAB2 to the level obtained by the TPA-conditioned medium, suggesting that the putative secreted DAB2 regulator is altered by STI571 qualitatively or quantitatively. Based on the fact that PDGF is present in the TPA-conditioned medium and STI571 is a potent PDGFR inhibitor, we quantify the amount of PDGF-BB, a ligand that activates both PDGFRa and PDGFRb, in the conditioned medium. An approximate 44% decrease in PDGF-BB was observed for the TPA/STI571-treated cells (86 pg/ml), in comparison to the TPA-treated cells (153 pg/ml) (Fig. 3B). The decrease in PDGF was not due to the change of PDGF gene expression, because STI571 did not alter PDGF-A and PDGF-B mRNA expression (data not shown). These results indicate that STI571 modulates the quantity of PDGF in the conditioned medium during megakaryocytic differentiation. 3.4. PDGF autocrine signaling regulates DAB2 expression To elicit whether there is a link between PDGF autocrine signaling and DAB2 expression, the anti-PDGF neutralization antibody that recognizes PDGF A and B chains and blocks PDGF receptor activation was added into the TPA-conditioned medium. Western blot analysis revealed that, when applying to the exponential growing K562 cells, this type of conditioned medium attenuated its effect on DAB2 induction in a dose-dependent manner (Fig. 4A). As a control, the IgG control antibody had no effect on DAB2 expression. To
C.-P. Tseng et al. / FEBS Letters 579 (2005) 4395–4401
Fig. 3. STI571 inhibits DAB2 induction and PDGF secretion. K562 cells were treated with ethanol (E, 0.01%), TPA (T, 10 ng/ml) or TPA plus STI571 (T + S) for 72 h. The conditioned medium was collected and applied to the exponential growing K562 cells for 24 and 48 h. Total cell lysates were subjected to Western blot analysis with anti-p96 (DAB2) and anti-b-actin antibodies (panel A). On the other hand, the supernatants were collected at 72 h after culture and the concentration of PDGF in the culture medium was quantified by the human PDGFBB immunoassay (panel B). The data represent the mean ± S.E. of three assays in a representative experiment. Similar results were obtained in two independent experiments.
demonstrate that PDGF signaling is modulated by the neutralization antibody, we used anti-phospho-MAPK antibody to analyze one of the PDGF downstream effectors, MAPK activation [21]. As shown in Fig. 4B, MAPK activation is inhibited by the PDGF neutralization antibody but not the IgG control antibody, indicating the blockage of PDGF signaling. To confirm the involvement of PDGF signaling in the regulation of DAB2 expression, the K627A (am) and truncated (at) forms of dominant negative PDGFRa and K634A (bm) form of dominant negative PDGFRb were used in the following study. These constructs have been shown previously as a
Fig. 4. PDGF neutralization antibody inhibits DAB2 expression. A and B. TPA-conditioned medium from K562 cells was preincubated with the indicated concentrations of PDGF neutralization antibody or IgG control antibody for 30 min. Then exponential growing K562 cells (8 · 104/ml) were cultured with the pre-treated conditioned medium for 24 h. Total cell extracts were collected and subjected to Western blot analysis with anti-p96 (DAB2) (panel A), anti-phospho-p42/p44MAPK and anti-p42/p44-MAPK (panel B) antibodies. The expression of b-actin was used as the control of equal protein loading.
C.-P. Tseng et al. / FEBS Letters 579 (2005) 4395–4401
powerful tool to address cell signaling mediated by the respective PDGFR [21,22]. Transient transfection of these plasmids increased in PDGFR expression, most prominent in TPA-treated cells, as demonstrated by Western blot analysis with the anti-PDGFRa and anti-PDGFRb antibodies (Fig. 5A). With approximate 50% transfection efficiency, TPA-mediated DAB2 induction was diminished by cotransfection of bm with either am or at (Fig. 5B). As a control, the cells transfected with wild-type PDGFRa (awt) and PDGFRb (bwt) had comparable DAB2 expression as the vector-transfected cells. Using the K562 stable cell lines expressing at or am, we found that blockage of PDGFRa alone is sufficient to inhibit TPA-mediated DAB2 induction (Fig. 5C). These data reveal the critical role of PDGF signaling in DAB2 expression during megakaryocytic differentiation of K562 cells. To further elucidate that DAB2 expression is regulated by the autocrine PDGF signaling, we transiently transfected K562 cells with pSecTag2A-PDGFB that produced secreted recombinant PDGF-BB due to the fusion of a murine Igjchain V-J2-C signal peptide at the N-terminus of PDGF-B
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cDNA sequences. Accordingly, we detected a significant increase in PDGF-BB (112 pg/ml) in the culture medium. Of these transfected cells, DAB2 was upregulated simultaneously with the increase in PDGF-BB. The induction of DAB2 was more prominent when K562 cells were treated with TPA (Fig. 5D). The PDGF-mediated DAB2 up-regulation was also observed in an oral cancer, non-hematopoietic cell line OECM1, indicating that the effect of PDGF on DAB2 gene is not cell type-specific. Together, these data strengthen the notion that PDGF is a positive regulator of DAB2 gene expression.
4. Discussion During megakaryocytic differentiation of K562 cells, both PDGF-A and PDGF-B polypeptides are induced by TPA through gene activation. Following secretion into the medium, PDGF-A and PDGF-B form homodimer or heterodimer and elicit autocrine effects on the culture cells through binding and activation of the membrane protein tyrosine kinase
Fig. 5. PDGF autocrine signaling regulates DAB2 expression. A. Expression levels of various PDGFRa and PDGFRb constructs. The vector control (V), wild type PDGFRa (awt) and PDGFRb (bwt), and dominant negative PDGFRa (am and at) and PDGFRb (bm) expression plasmids (6 lg) were transiently transfected into K562 cells (2 · 106). After the indicated treatment for 24 h, total cell lysates were collected and subjected to Western blot analyses with the anti-PDGFRa and anti-PDGFRb antibodies, respectively. B and C. Dominant negative PDGFR reduced TPAmediated DAB2 expression. K562 cells were transiently transfected with pcDNA3 vector control, awt + bwt, am + bm, and at + bm. These transfected cells (panel B) and K562 cells stably expressing pcDNA3 vector, am, and at (panel C) were treated with TPA (T, 10 ng/ml) or solvent control ethanol (E, 0.01%), respectively. Total cell lysates were then collected and subjected to Western blot analyses with anti-p96 (DAB2) or anti-PDGFRa antibodies. D. PDGF up-regulates DAB2 expression. The vector control or the PDGF-B expression plasmid pSec-Tag2A-PDGFB was transiently transfected into K562 and OEC-M1 cells. At 48 h after the indicated treatment, cell lysates were collected and subjected to Western blot analysis with anti-p96 (DAB2) antibody. The expression of b-actin was used as the control of equal protein loading for each assay.
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receptors PDGFRa and b [1]. The signals are then propagated through PI3 kinase, Ras/MAPK, and PLCc pathways that eventually result in a biological response to affect cell proliferation and differentiation [23]. In this study, PDGF is unveiled for the first time as a novel DAB2 regulator. This conclusion is supported by the findings that PDGF neutralization antibody, PDGFR inhibitor STI571 and dominant negative PDGFR could interfere PDGF signaling and inhibit TPA- and conditioned medium-mediated DAB2 upregulation. To further support this notion, we found that expression of recombinant PDGF augments DAB2 induction in megakaryocytic differentiating cells. Multiple levels of regulation are involved in the control of DAB2 expression. Activation of p42/p44-MAPK regulates DAB2 transcriptional activity during megakaryocytic differentiation [16]. In visceral endoderm and gastrointestinal development, the transcription factors GATA-4, GATA-5, and GATA-6 are the primary positive regulators for DAB2 expression [17,18]. In macrophage, PU.1 and interferon consensus sequence binding protein are the positive and negative regulator of DAB2 transcription, respectively [14]. Through microarray analysis, DAB2 is identified as a target gene of RARb2 during retinoic acid-induced differentiation of F9 cells [19], VHL in the hypoxic response pathway [24], and GATA-3 during mouse embryonic development [25]. In this study, interference of PDGF signaling has only moderate effect on DAB2 mRNA expression and promoter activity, implicating that PDGF may elicit its activity at the post-transcriptional or translational level. Hence, PDGF-mediated cell signaling and regulation represent an additional mechanism for controlling DAB2 expression during megakaryocytic differentiation. Such means of regulation may augment the level of protein expression and sustain DAB2 expression in the megakaryocytic differentiating cells. Similar regulatory mechanism has been shown previously for the expression of SCL, AML1/RUNX1 and prostaglandin endoperoxide H synthase-1 proteins associated with megakaryocytic differentiation [26–28]. It is noted that the majority of those mRNAs under translational regulation have long 5 0 untranslated regions (UTR) with structured regions which inhibit the scanning mechanisms of translation. Prediction of the DAB2 mRNA 5 0 -UTR folding structure with the RNA mfold version 2.3 server software from the Bioinformatic Center at Rensselaer and Wadsworth [29] reveals three obvious stem-loops with a significant GC pairing. Whether these stem-loop structures are important in the translational regulation of DAB2 by PDGF requires further analysis. Apart from the effects on DAB2, the receptor protein tyrosine kinase inhibitor STI571 also inhibits megakaryocytic differentiation. Previous study has demonstrated that STI571 affected the development and function of dendritic cells generated from CD34+ peripheral blood progenitor cells [30]. The in vitro development of the monocyte/macrophage lineage from normal human bone marrow progenitors was also inhibited by STI571, whereas neutrophil and eosinophil development was less affected [31]. The STI571-induced apoptosis and the inhibition of c-Kit activity did not account for its effect on cell differentiation [30,31]. STI571 may therefore have an inhibitory activity for other kinase(s) that play a role in hematopoietic cell differentiation. We reveal in this study that inhibition of PDGF signaling accounts for the effect of STI571 on megakaryocytic differentiation. These results confirm previous observation that recombinant PDGF significantly enhances megakaryocyte col-
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ony formation in a dose-dependent manner [32] and further implicates a role of PDGF in megakaryocytopoiesis. In summary, this study shows for the first time that PDGF autocrine signaling is a positive regulator for DAB2 expression and megakaryocytic differentiation. The present study further suggests that there is interplay between DAB2 expression, MAPK activation, autocrine PDGF signaling and megakaryocytopoiesis. Apparently, such information leads us for a better understanding of how DAB2 is regulated during megakaryocytic differentiation. Acknowledgments: This work was supported in part by the National Science Council Grant NSC 93-2314-B-182-072 and NSC 93-2745B182-006-URD to C.P.T.
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C.-P. Tseng et al. / FEBS Letters 579 (2005) 4395–4401 [15] Huang, C.L., Cheng, J.C., Liao, C.H., Stern, A., Hsieh, J.T., Wang, C.H., Hsu, H.L. and Tseng, C.P. (2004) Disabled-2 is a negative regulator of integrin a IIbb 3-mediated fibrinogen adhesion and cell signaling. J. Biol. Chem. 279, 42279–42289. [16] Tseng, C.P., Huang, C.H., Tseng, C.C., Lin, M.H., Hsieh, J.T. and Tseng, C.H. (2001) Induction of Disabled-2 gene during megakaryocyte differentiation of K562 cells. Biochem. Biophys. Res. Commun. 285, 129–135. [17] Akiyama, Y., Watkins, N., Suzuki, H., Jair, K.W., van Engeland, M., Esteller, M., Sakai, H., Ren, C.Y., Yuasa, Y., Herman, J.G. and Baylin, S.B. (2003) GATA-4 and GATA-5 transcription factor genes and potential downstream antitumor target genes are epigenetically silenced in colorectal and gastric cancer. Mol. Cell. Biol. 23, 8429–8439. [18] Morrisey, E.E., Musco, S., Chen, M.Y., Lu, M.M., Leiden, J.M. and Parmacek, M.S. (2000) The gene encoding the mitogenresponsive phosphoprotein Dab2 is differentially regulated by GATA-6 and GATA-4 in the visceral endoderm. J. Biol. Chem. 275, 19949–19954. [19] Zhuang, Y., Faria, T.N., Chambon, P. and Gudas, L.J. (2003) Identification and characterization of retinoic acid receptor beta2 target genes in F9 teratocarinoma cells. Mol. Cancer Res. 1, 619– 630. [20] Buchdunger, E., Cioffi, C.L., Law, N., Stover, D., Ohno-Jones, S., Druker, B.J. and Lydon, N.B. (2000) Abl protein-tyrosine kinase inhibitor STI571 inhibits in vitro signal transduction mediated by c-kit and platelet-derived growth factor receptors. J. Pharmacol. Exp. Ther. 295, 139–145. [21] Emaduddin, M., Ekman, S., Ronnstrand, L. and Heldin, C.H. (1999) Functional co-operation between the subunits in heterodimeric platelet-derived growth factor receptor complexes. Biochem. J. 341, 523–528. [22] Ikuno, Y. and Kazlauskas, A. (2002) An in vivo gene therapy approach for experimental proliferative vitreoretinopathy using the truncated platelet-derived growth factor alpha receptor. Invest. Ophthalmol. Vis. Sci. 43, 2406–2411. [23] Claesson-Welsh, L. (1996) Mechanism of action of plateletderived growth factor. Int. J. Biochem. Cell Biol. 28, 373–385.
4401 [24] Maina, E.N., Morris, M.R., Zatyka, M., Raval, R.R., Banks, R.E., Richards, F.M., Johnson, C.M. and Maher, E.R. (2005) Identification of novel VHL target genes and relationship to hypoxic response pathways. Oncogene 24, 4549–4558. [25] Airik, R., Karner, M., Karis, A. and Karner, J. (2005) Gene expression analysis of Gata3-/- mice by using cDNA microarray technology. Life Sci. 76, 2559–2568. [26] Calkhoven, C.F., Muller, C., Martin, R., Krosl, G., Pietsch, H., Hoang, T. and Leutz, A. (2003) Translational control of SCLisoform expression in hematopoietic lineage choice. Genes Dev. 17, 959–964. [27] Pozner, A., Goldenberg, D., Negreanu, V., Le, S.Y., Elroy-Stein, O., Levanon, D. and Groner, Y. (2000) Transcription-coupled translation control of AML1/RUNX1 is mediated by cap- and internal ribosome entry site-dependent mechanisms. Mol. Cell. Biol. 20, 2297–2307. [28] Duquette, M. and Laneuville, O. (2002) Translational regulation of prostaglandin endoperoxide H synthase-1 mRNA in megakaryocytic MEG-01 cells. Specific protein binding to a conserved 20-nucleotide CIS element in the 3 0 -untranslated region. J. Biol. Chem. 277, 44631–44637. [29] Mathews, D.H., Sabina, J., Zuker, M. and Turner, D.H. (1999) Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J. Mol. Biol. 288, 911–940. [30] Appel, S., Boehmler, A.M., Grunebach, F., Muller, M.R., Rupf, A., Weck, M.M., Hartmann, U., Reichardt, V.L., Kanz, L., Brummendorf, T.H. and Brossart, P. (2004) Imatinib mesylate affects the development and function of dendritic cells generated from CD34+ peripheral blood progenitor cells. Blood 103, 538– 544. [31] Dewar, A.L., Domaschenz, R.M., Doherty, K.V., Hughes, T.P. and Lyons, A.B. (2003) Imatinib inhibits the in vitro development of the monocyte/macrophage lineage from normal human bone marrow progenitors. Leukemia 17, 1713–1721. [32] Yang, M., Chesterman, C.N. and Chong, B.H. (1995) Recombinant PDGF enhances megakaryocytopoiesis in vitro. Br. J. Haematol. 91, 285–289.
FEBS Letters 579 (2005) 4395–4401
Autocrine signaling of platelet-derived growth factor regulates disabled-2 expression during megakaryocytic differentiation of K562 cells Ching-Ping Tsenga,b,*, Pam Changa, Chien-Ling Huanga, Ju-Chien Chengc, Shy-Shin Changd b
a Graduate Institute of Basic Medical Sciences, Chang Gung University, Tao-Yuan 333, Taiwan, ROC Graduate Institute of Medical Biotechnology, Chang Gung University, 259 Wei-Hwa 1st Road, Kwei-Shan, Tao-Yuan 333, Taiwan, ROC c School of Medical Laboratory Sciences and Biotechnology, China Medical University, Taichung 404, Taiwan, ROC d Department of Emergency Medicine, Chang Gung Memorial Hospital, Tao-Yuan 333, Taiwan, ROC
Received 17 January 2005; revised 25 June 2005; accepted 27 June 2005 Available online 19 July 2005 Edited by Veli-Pekka Lehto
Abstract Platelet-derived growth factor (PDGF) is involved in megakaryocytopoiesis and is secreted into the culture medium during megakaryocytic differentiation of human leukemic cells. We investigate whether PDGF plays a role in the regulation of the adapter protein Disabled-2 (DAB2) that expresses abundantly in platelets and megakaryocytes. Western blot analysis revealed that conditioned medium from 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated, megakaryocytic differentiating K562 cells upregulated DAB2 expression. DAB2 induction and megakaryocytic differentiation was abrogated when cells were co-treated with the PDGF receptor inhibitor STI571 or when the conditioned medium was derived from TPA-plus STI571treated cells. Although the level of PDGF mRNA was not altered by STI571, an approximate 44% decrease in PDGF in the conditioned medium was observed. Consistent with these findings, interfering PDGF signaling by PDGF neutralization antibody or dominant negative PDGF receptors attenuated DAB2 expression. Accordingly, transfection of an expression plasmid encoding secreted PDGF upregulated DAB2. This study shows for the first time that PDGF autocrine signaling regulates DAB2 expression during megakaryocytic differentiation. 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Disabled-2; Megakaryocytic differentiation; Platelet-derived growth factor; STI571
1. Introduction Platelet-derived growth factor (PDGF) constitutes a family of peptide growth factors that includes at least four dimeric forms, PDGF-AA, -AB, -BB, and -CC [1]. PDGF dimer is linked together by disulfide bonds and activates two specific receptors, the PDGF a-receptor (PDGFRa) and the PDGF b-receptor (PDGFRb). In blood, PDGF expresses abundantly in megakaryocyte and platelet a-granule. Targeting disruption of PDGF-B or PDGFRb results in various hematopoietic defects, including microvessel leakage, hemorrhage, microaneurysm formation, anemia, and thrombocytopenia [2,3]. Apart
from its pivotal role in thrombosis and hemostasis, PDGF is a positive regulator for megakaryocytopoiesis. During megakaryocytic differentiation of human leukemic cells, PDGF-A and PDGF-B are induced through gene activation and are secreted into the culture medium together with other autocrine regulatory factors [4,5]. The conditioned medium retains the ability to induce megakaryocytic differentiation [6], with PDGF signaling mediates the expression of megakaryocyte-associated transcription factors NF-E2, GATA-1, and c-FOS [7]. PDGF is thus considered as one of the megakaryocytic lineage determination factors present in the conditioned medium. Disabled-2 (DAB2) is an adapter protein that is implicated in growth factor signaling [8,9], endocytosis [10–12], and cell adhesive function [13,14]. During in vitro megakaryocytic differentiation of human leukemic cells and human umbilical cord blood CD34+ hematopoietic stem cells, DAB2 is upregulated and acts as a negative regulator in integrin a IIbb3-mediated fibrinogen adhesion and cell signaling [13,15,16]. DAB2 is also induced during visceral endoderm and gastrointestinal development and retinoic acid-induced F9 cell differentiation [17–19]. Of these differentiation models, DAB2 is mainly regulated at the transcriptional level. However, our previous study [16] in comparing the extent of DAB2 mRNA and protein expression during megakaryocytic differentiation of K562 cells implicates the presence of an additional level of regulation in the control of DAB2 gene expression that has not yet been characterized. With the tight association between DAB2 expression and megakaryocytic differentiation and the presence of megakaryocytic lineage determination factors in the conditioned medium, we address whether the autocrine regulatory factors plays a role in DAB2 expression. In this study, we report that the conditioned medium from megakaryocytic differentiating cells retains the ability to induce DAB2 and implicates a link between DAB2 expression and autocrine signaling. Through molecular targeting approaches using PDGF receptor inhibitor STI571 [20], dominant negative PDGF receptors and expression of recombinant PDGF, we further demonstrate that the autocrine signaling of PDGF plays a crucial role in the regulation of DAB2 expression mainly through a non-transcriptional control mechanism. 2. Materials and methods
*
Corresponding author. Fax: +886 03 2118355. E-mail address: [email protected] (C.-P. Tseng). Abbreviations: DAB2, Disabled-2; PDGF, platelet-derived growth factor; TPA, 12-O-tetradecanoylphorbol-13-acetate
2.1. Materials The anti-b-actin antibody and TPA were purchased from Sigma Chemical Co. (St. Louis, MO). The anti-DAB2 antibody p96 was purchased from BD Transduction Laboratories (San Jose, CA). The
0014-5793/$30.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.06.080
4396 anti-b3, CD61, PDGFRa, and PDGFRb antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-p42/p44 MAPK and phospho-p42/p44 MAPK antibodies were purchased from New England Biolab (Beverly, MA). Quantikine human PDGF-BB immunoassay and the anti-PDGF neutralization antibody were from R&D Systems Inc. (Minneapolis, MN). The STI571 (Gleevec) is the product of Novartis Pharma AG (Basel, Switzerland). The 2· SYBR Green I PCR master mix was purchased from Applied Biosystems (Foster City, CA). The kinase-inactive K627A PDGFRa and K634A PDGFRb expression plasmids [21] were a kind gift from Dr. CarlHenrik Heldin (Ludwig Institute for Cancer Research, Uppsala, Sweden). The truncated PDGFRa expression plasmid [22] was a kind gift from Dr. Andrius Kazlauskas (The Schepens Eye Research Institute, Harvard Medical School, Boston, MA). 2.2. Plasmid construction To generate pcDNA3-D-PDGFRa, a 2.0-kb NotI–ApaI DNA fragment corresponding to the truncated PDGFRa in pLXSN vector [22] was subcloned into the pcDNA3 expression vector. To construct pSecTag2A-PDGFB, PDGF-B cDNA was obtained from K562 cells by RT-PCR using the primer set PDGFB-F (5 0 -AAGCTTTCTGGGTTCCCTGACCAT-3 0 ) and PDGFB-R (5 0 -GGATCCTCGAGGCTCCAAGGGT-3 0 ). The full-length PDGF-B cDNA was then subcloned into the BamHI–HindIII sites of the pSecTag2A expression vector (Invitrogen). 2.3. Cell culture, conditioned medium collection, transient transfection, and Western blot analysis K562 and OEC-M1 cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and antibiotics. The conditioned medium was obtained from the supernatant of K562 cells with the indicated treatment after centrifugation at 1500 rpm for 5 min. Transient transfection, preparation of total cell lysates, and Western blot analysis were performed as described previously [13]. 2.4. Analysis of CD61 expression by flow cytometry K562 cells were collected and washed by FABS buffer (1· PBS, 1% FBS, and 0.1% NaN3). The CD61 mouse monoclonal antibody (2 lg/ ml) was added to the cell suspension followed by incubation with fluorescein isothiocyanate-conjugated goat anti-mouse antibody (0.4 lg/ml). After several washes with the FABS buffer, K562 cells were subjected to flow cytometric analysis with FACSCAN and the data were analyzed using the CellQuest software (Becton Dickinson, San Jose, CA).
C.-P. Tseng et al. / FEBS Letters 579 (2005) 4395–4401 assay diluent buffer. The mixture was then added into a 96-wells plate that was pre-coated with PDGF-Rb/Fc chimera. Following incubation with the substrate solution, the stop solution was added and the OD450 was determined using a microplate reader. 2.8. Neutralization of PDGF activity The PDGF activity in the conditioned medium was neutralized by incubating the medium with anti-PDGF neutralization antibody or control IgG antibody for 30 min. The medium was then used to culture exponential growing K562 cells to determine the effect of PDGF on DAB2. 2.9. Establishment of dominant negative PDGFRa stable cell lines K562 cells were transfected with 6 lg of the kinase-inactive K627A PDGFRa or the truncated PDGFRa (pcDNA3-D-PDGFRa) expression plasmids. After neomycin selection (400 lg/ml) for 1 month, the stable cell lines expressing kinase-inactive or truncated PDGFRa were identified by Western blot analysis using anti-PDGFRa antibody.
3. Results 3.1. TPA-conditioned medium induces DAB2 expression and megakaryocytic differentiation Previous studies have shown that the conditioned medium from TPA-treated K562 cells mimics the effect of TPA and induces integrin b3 expression and morphological changes associated with megakaryocytic differentiation. To determine whether the conditioned medium modulates DAB2 expression, 1–3 days TPA-conditioned medium was collected and applied to the exponential growing K562 cells. Western blot analysis revealed that DAB2 was induced with maximum induction by the medium that has been ‘‘conditioned’’ for 72 h (Fig. 1A). The cells also demonstrated a typical differentiation phenotype with the characteristics of cell enlargement, decrease in N/C ratio, and nucleus with multiple lobes (data not shown). As a control, the conditioned medium derived from the cells treated with solvent control ethanol had no effect on DAB2 expression and megakaryocytic differentiation.
2.5. Real-time quantitative RT-PCR The one-step real-time quantitative RT-PCR was performed using the GenAmp 5700 Sequence Detection System (Applied Biosystems, Foster City, CA). Briefly, 300 ng of total RNA was added into a reaction mixture containing 25 ll of 2· SYBR Green I PCR master mix, 1.25 ll of 40· reverse transcriptase mixture, 1 ll of forward primer Dab2-F (100 nM, 5 0 -AGTGAGGCCCTAAGGATTCTAGATGACCAAACTA-3 0 ), and 1 ll of reverse primer Dab2-R (100 nM, 5 0 -CAGGATATCTTTGCTTTCTGTTGGACTATTTAGGTC-3 0 ) in a final volume of 50 ll. The condition for RT-PCR was 1 cycle of 48 C for 30 min, 1 cycle of 95 C for 10 min, and 40 cycles of 95 C for 15 s and 60 C for 1 min. The cycle threshold value (CT) was used to assess the quantity of target gene. The relative fold difference for the expression of DAB2 mRNA was normalized by the expression of b-actin and was determined using the value of 2DDCT. 2.6. Luciferase activity assay The K562 cells (4 · 105) were transiently transfected with 200 ng of DAB2 promoter firefly luciferase reporter construct phDab2-luc [16] and 2 ng of Renilla luciferase expression plasmid pRL-TK. After indicated treatment, the cell lysates were subjected to luciferase activity assay according to the manufacturerÕs instructions. The firefly luciferase activity was normalized by the Renilla luciferase activity in each individual sample. 2.7. PDGF quantification The human PDGF-BB immunoassay (R&D Systems Inc.) was used to quantify the amount of PDGF in the conditioned medium. Briefly, 100 ll of PDGF standard or sample medium was mixed with 100 ll of
Fig. 1. TPA-conditioned medium induces DAB2 expression. (A,B) K562 cells (5 · 104/ml) were treated with TPA (T, 10 ng/ml) or solvent control ethanol (E) for the indicated time (panel A). Alternatively, K562 cells were incubated with E or T for the indicated time and the medium was replaced by fresh RPMI 1640 medium without TPA for a total of 72 h (panel B). Conditioned medium was then collected and applied to exponential growing K562 cells (1.5 · 104/ml) for 48 h. Total cell lysates were collected and subjected to Western blot analysis with anti-p96 (DAB2) antibody. The expression of b-actin was used as a control for equal protein loading. Essentially similar results were obtained in 3 independent experiments.
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To eliminate the concern that DAB2 induction is due to TPA carryover, we modified the way for conditioned medium preparation in the following experiment. At 6, 12, and 24 h after TPA treatment, the medium was removed and replaced by fresh medium without TPA. The conditioned medium was then collected at 72 h after initial TPA treatment and applied to exponential growing K562 cells. Western blot analysis revealed that DAB2 was induced to the level comparable to the medium that has been conditioned for 72 h (e.g., treatment for 72 h without removal of TPA) (Fig. 1B). These results rule out TPA carryover as the cause of DAB2 induction and implicate the presence of soluble DAB2 regulator(s) in the TPA-conditioned medium.
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3.2. STI571 inhibits TPA- and conditioned medium-induced DAB2 expression and megakaryocytic differentiation PDGF is involved in megakaryocytopoiesis and is secreted into the culture medium during megakaryocytic differentiation of human leukemic cells. We co-treated K562 cells with TPA and PDGF signaling inhibitor STI571 to investigate whether PDGF plays a role in the regulation of DAB2. At the concentrations between 0.01 and 1 lM, STI571 inhibited DAB2 expression in a dose-dependent manner. More than 80% of DAB2 expression was inhibited by 1 lM of STI571 in both K562 cells (Fig. 2A) and megakaryocytic cell line HEL (data not shown). The conditioned medium-induced DAB2 expression was also suppressed by STI571 (Fig. 2B). Because the
Fig. 2. STI571 inhibits DAB2 expression and megakaryocytic differentiation. (A) STI571 inhibits DAB2 and integrin b3 expression in a dosedependent manner. K562 cells (5 · 104/ml) were pretreated with the indicated concentrations of STI571 for 15 min followed by 48 h treatment with TPA (T, 10 ng/ml) or solvent control ethanol (E, 0.01%). Total cell lysates were subjected to Western blot analysis with anti-p96 (DAB2) and antiintegrin b3 antibodies. The expression of b-actin was used as a control for equal protein loading. B. STI571 inhibits conditioned medium-induced DAB2 expression. Either general K562 cell culture medium or TPA-conditioned medium was collected and applied to exponential growing K562 cells for 72 h, in the presence of 100 nM STI571 (S), 10 ng/ml TPA (T), or solvent vehicle control (E). Total cell lysates were collected and subjected to Western blot analysis with anti-p96 (DAB2) and anti-b-actin antibodies. C. Effect of STI571 on TPA-induced DAB2 mRNA expression. The K562 cells (5 · 104/ml) were treated with vehicle ethanol, TPA, or TPA + STI571 (100 nM) for 48 h. The expression level of DAB2 mRNA was determined by real-time RT-PCR using 300 ng total RNA and was normalized by the levels of b-actin mRNA. D. STI571 does not inhibit TPA-induced DAB2 promoter activity. The K562 cells (4 · 105) were transfected with phDAB2-luc (200 ng) and the Renilla luciferase expression plasmid pRL-TK (2 ng) by the DMRIE-C transfection reagent. After treatment with ethanol (0.01%), TPA (10 ng/ml), and TPA + STI571 (100 nM) for 24 h, the cell lysates were collected for luciferase activity assay using the dual-luciferase reporter assay system. The DAB2 promoter activity was normalized by the Renilla luciferase activity and the normalized activity for the ethanol-treated control group was arbitrary set as 1. The data represent the mean ± S.E. of three assays in a representative experiment. Similar results were obtained in three independent experiments. E and F. Effects of STI571 on TPA-induced CD61 expression and cell morphology. K562 cells were treated with ethanol, TPA, and TPA plus STI571 for 48 h. Flow cytometric analysis was performed to detect the megakaryocytic differentiation surface marker CD61 (panel E). Aliquots of the cell suspension were cytospin, stained with Liu stain reagents, and observed by light microscopy. A typical field was shown for K562 cells treated with ethanol (control), TPA, and TPA + STI571, respectively (panel F).
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survival rate for the cells treated with STI571 was more than 95%, it ruled out non-specific cellular toxicity as the cause for the decrease in DAB2. Whether STI571 affects DAB2 transcription was determined by both real-time RT-PCR and promoter activity analysis. TPA induced a 12-fold increase in DAB2 mRNA. In the presence of STI571, a moderate 30% decrease in TPA-induced DAB2 mRNA was observed (Fig. 2C). To confirm this finding, we transfected phDab2-luc, a TPA-responsive DAB2 promoter luciferase reporter construct, into K562 cells followed by the indicated treatment (Fig. 2D). Consistent with the results of real-time RT-PCR, TPA induced approximate 15fold increase in phDab2-luc activity and the induction was not significantly altered by STI571. With the distinct extent of inhibition in DAB2 mRNA and protein, these data implicate that post-transcriptional control may be involved in STI571-mediated inhibition of DAB2 expression. In addition to modulate DAB2, integrin b3 expression was also diminished by STI571 (Fig. 2A). To elucidate the effect of STI571 on megakaryocytic differentiation, flow cytometric analysis was performed to detect the expression of megakaryocytic differentiation surface marker CD61. Our results revealed that CD61 was reduced to 23% for TPA/STI571-treated cells compared to 40% for TPA-treated cells (Fig. 2E). Accordingly, the typical phenotypes of megakaryocytic differentiation were less prominent in the TPA/STI571-treated cells (Fig. 2F). These results indicate that sub-apoptotic concentration of STI571 also impedes megakaryocytic differentiation of K562 cells. 3.3. STI571 reduces PDGF secretion into the culture medium To investigate whether STI571 mediates its effect by modulating autocrine signaling, the conditioned medium from TPA/STI571-treated cells was collected at 72 h after treatment and applied to the exponential growing K562 cells. As shown in Fig. 3A, this type of conditioned medium did not induce DAB2 to the level obtained by the TPA-conditioned medium, suggesting that the putative secreted DAB2 regulator is altered by STI571 qualitatively or quantitatively. Based on the fact that PDGF is present in the TPA-conditioned medium and STI571 is a potent PDGFR inhibitor, we quantify the amount of PDGF-BB, a ligand that activates both PDGFRa and PDGFRb, in the conditioned medium. An approximate 44% decrease in PDGF-BB was observed for the TPA/STI571-treated cells (86 pg/ml), in comparison to the TPA-treated cells (153 pg/ml) (Fig. 3B). The decrease in PDGF was not due to the change of PDGF gene expression, because STI571 did not alter PDGF-A and PDGF-B mRNA expression (data not shown). These results indicate that STI571 modulates the quantity of PDGF in the conditioned medium during megakaryocytic differentiation. 3.4. PDGF autocrine signaling regulates DAB2 expression To elicit whether there is a link between PDGF autocrine signaling and DAB2 expression, the anti-PDGF neutralization antibody that recognizes PDGF A and B chains and blocks PDGF receptor activation was added into the TPA-conditioned medium. Western blot analysis revealed that, when applying to the exponential growing K562 cells, this type of conditioned medium attenuated its effect on DAB2 induction in a dose-dependent manner (Fig. 4A). As a control, the IgG control antibody had no effect on DAB2 expression. To
C.-P. Tseng et al. / FEBS Letters 579 (2005) 4395–4401
Fig. 3. STI571 inhibits DAB2 induction and PDGF secretion. K562 cells were treated with ethanol (E, 0.01%), TPA (T, 10 ng/ml) or TPA plus STI571 (T + S) for 72 h. The conditioned medium was collected and applied to the exponential growing K562 cells for 24 and 48 h. Total cell lysates were subjected to Western blot analysis with anti-p96 (DAB2) and anti-b-actin antibodies (panel A). On the other hand, the supernatants were collected at 72 h after culture and the concentration of PDGF in the culture medium was quantified by the human PDGFBB immunoassay (panel B). The data represent the mean ± S.E. of three assays in a representative experiment. Similar results were obtained in two independent experiments.
demonstrate that PDGF signaling is modulated by the neutralization antibody, we used anti-phospho-MAPK antibody to analyze one of the PDGF downstream effectors, MAPK activation [21]. As shown in Fig. 4B, MAPK activation is inhibited by the PDGF neutralization antibody but not the IgG control antibody, indicating the blockage of PDGF signaling. To confirm the involvement of PDGF signaling in the regulation of DAB2 expression, the K627A (am) and truncated (at) forms of dominant negative PDGFRa and K634A (bm) form of dominant negative PDGFRb were used in the following study. These constructs have been shown previously as a
Fig. 4. PDGF neutralization antibody inhibits DAB2 expression. A and B. TPA-conditioned medium from K562 cells was preincubated with the indicated concentrations of PDGF neutralization antibody or IgG control antibody for 30 min. Then exponential growing K562 cells (8 · 104/ml) were cultured with the pre-treated conditioned medium for 24 h. Total cell extracts were collected and subjected to Western blot analysis with anti-p96 (DAB2) (panel A), anti-phospho-p42/p44MAPK and anti-p42/p44-MAPK (panel B) antibodies. The expression of b-actin was used as the control of equal protein loading.
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powerful tool to address cell signaling mediated by the respective PDGFR [21,22]. Transient transfection of these plasmids increased in PDGFR expression, most prominent in TPA-treated cells, as demonstrated by Western blot analysis with the anti-PDGFRa and anti-PDGFRb antibodies (Fig. 5A). With approximate 50% transfection efficiency, TPA-mediated DAB2 induction was diminished by cotransfection of bm with either am or at (Fig. 5B). As a control, the cells transfected with wild-type PDGFRa (awt) and PDGFRb (bwt) had comparable DAB2 expression as the vector-transfected cells. Using the K562 stable cell lines expressing at or am, we found that blockage of PDGFRa alone is sufficient to inhibit TPA-mediated DAB2 induction (Fig. 5C). These data reveal the critical role of PDGF signaling in DAB2 expression during megakaryocytic differentiation of K562 cells. To further elucidate that DAB2 expression is regulated by the autocrine PDGF signaling, we transiently transfected K562 cells with pSecTag2A-PDGFB that produced secreted recombinant PDGF-BB due to the fusion of a murine Igjchain V-J2-C signal peptide at the N-terminus of PDGF-B
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cDNA sequences. Accordingly, we detected a significant increase in PDGF-BB (112 pg/ml) in the culture medium. Of these transfected cells, DAB2 was upregulated simultaneously with the increase in PDGF-BB. The induction of DAB2 was more prominent when K562 cells were treated with TPA (Fig. 5D). The PDGF-mediated DAB2 up-regulation was also observed in an oral cancer, non-hematopoietic cell line OECM1, indicating that the effect of PDGF on DAB2 gene is not cell type-specific. Together, these data strengthen the notion that PDGF is a positive regulator of DAB2 gene expression.
4. Discussion During megakaryocytic differentiation of K562 cells, both PDGF-A and PDGF-B polypeptides are induced by TPA through gene activation. Following secretion into the medium, PDGF-A and PDGF-B form homodimer or heterodimer and elicit autocrine effects on the culture cells through binding and activation of the membrane protein tyrosine kinase
Fig. 5. PDGF autocrine signaling regulates DAB2 expression. A. Expression levels of various PDGFRa and PDGFRb constructs. The vector control (V), wild type PDGFRa (awt) and PDGFRb (bwt), and dominant negative PDGFRa (am and at) and PDGFRb (bm) expression plasmids (6 lg) were transiently transfected into K562 cells (2 · 106). After the indicated treatment for 24 h, total cell lysates were collected and subjected to Western blot analyses with the anti-PDGFRa and anti-PDGFRb antibodies, respectively. B and C. Dominant negative PDGFR reduced TPAmediated DAB2 expression. K562 cells were transiently transfected with pcDNA3 vector control, awt + bwt, am + bm, and at + bm. These transfected cells (panel B) and K562 cells stably expressing pcDNA3 vector, am, and at (panel C) were treated with TPA (T, 10 ng/ml) or solvent control ethanol (E, 0.01%), respectively. Total cell lysates were then collected and subjected to Western blot analyses with anti-p96 (DAB2) or anti-PDGFRa antibodies. D. PDGF up-regulates DAB2 expression. The vector control or the PDGF-B expression plasmid pSec-Tag2A-PDGFB was transiently transfected into K562 and OEC-M1 cells. At 48 h after the indicated treatment, cell lysates were collected and subjected to Western blot analysis with anti-p96 (DAB2) antibody. The expression of b-actin was used as the control of equal protein loading for each assay.
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receptors PDGFRa and b [1]. The signals are then propagated through PI3 kinase, Ras/MAPK, and PLCc pathways that eventually result in a biological response to affect cell proliferation and differentiation [23]. In this study, PDGF is unveiled for the first time as a novel DAB2 regulator. This conclusion is supported by the findings that PDGF neutralization antibody, PDGFR inhibitor STI571 and dominant negative PDGFR could interfere PDGF signaling and inhibit TPA- and conditioned medium-mediated DAB2 upregulation. To further support this notion, we found that expression of recombinant PDGF augments DAB2 induction in megakaryocytic differentiating cells. Multiple levels of regulation are involved in the control of DAB2 expression. Activation of p42/p44-MAPK regulates DAB2 transcriptional activity during megakaryocytic differentiation [16]. In visceral endoderm and gastrointestinal development, the transcription factors GATA-4, GATA-5, and GATA-6 are the primary positive regulators for DAB2 expression [17,18]. In macrophage, PU.1 and interferon consensus sequence binding protein are the positive and negative regulator of DAB2 transcription, respectively [14]. Through microarray analysis, DAB2 is identified as a target gene of RARb2 during retinoic acid-induced differentiation of F9 cells [19], VHL in the hypoxic response pathway [24], and GATA-3 during mouse embryonic development [25]. In this study, interference of PDGF signaling has only moderate effect on DAB2 mRNA expression and promoter activity, implicating that PDGF may elicit its activity at the post-transcriptional or translational level. Hence, PDGF-mediated cell signaling and regulation represent an additional mechanism for controlling DAB2 expression during megakaryocytic differentiation. Such means of regulation may augment the level of protein expression and sustain DAB2 expression in the megakaryocytic differentiating cells. Similar regulatory mechanism has been shown previously for the expression of SCL, AML1/RUNX1 and prostaglandin endoperoxide H synthase-1 proteins associated with megakaryocytic differentiation [26–28]. It is noted that the majority of those mRNAs under translational regulation have long 5 0 untranslated regions (UTR) with structured regions which inhibit the scanning mechanisms of translation. Prediction of the DAB2 mRNA 5 0 -UTR folding structure with the RNA mfold version 2.3 server software from the Bioinformatic Center at Rensselaer and Wadsworth [29] reveals three obvious stem-loops with a significant GC pairing. Whether these stem-loop structures are important in the translational regulation of DAB2 by PDGF requires further analysis. Apart from the effects on DAB2, the receptor protein tyrosine kinase inhibitor STI571 also inhibits megakaryocytic differentiation. Previous study has demonstrated that STI571 affected the development and function of dendritic cells generated from CD34+ peripheral blood progenitor cells [30]. The in vitro development of the monocyte/macrophage lineage from normal human bone marrow progenitors was also inhibited by STI571, whereas neutrophil and eosinophil development was less affected [31]. The STI571-induced apoptosis and the inhibition of c-Kit activity did not account for its effect on cell differentiation [30,31]. STI571 may therefore have an inhibitory activity for other kinase(s) that play a role in hematopoietic cell differentiation. We reveal in this study that inhibition of PDGF signaling accounts for the effect of STI571 on megakaryocytic differentiation. These results confirm previous observation that recombinant PDGF significantly enhances megakaryocyte col-
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ony formation in a dose-dependent manner [32] and further implicates a role of PDGF in megakaryocytopoiesis. In summary, this study shows for the first time that PDGF autocrine signaling is a positive regulator for DAB2 expression and megakaryocytic differentiation. The present study further suggests that there is interplay between DAB2 expression, MAPK activation, autocrine PDGF signaling and megakaryocytopoiesis. Apparently, such information leads us for a better understanding of how DAB2 is regulated during megakaryocytic differentiation. Acknowledgments: This work was supported in part by the National Science Council Grant NSC 93-2314-B-182-072 and NSC 93-2745B182-006-URD to C.P.T.
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