PMA-induced Megakaryocytic Differentiation Research Articles

The involvement of krüppel-like transcription factor 2 in megakaryocytic differentiation induction by phorbol 12-myrestrat 13-acetate

BackgroundMegakaryocytic differentiation is a complicated process regulated by a series of transcription factors in a context- and stage-dependent manner. Recent studies have suggested that krüppel-like transcription factor 2 (KLF2) is involved in the control of embryonic erythroid precursor cell differentiation and maturation. However, the function and mechanism of KLF2 in regulating megakaryocytic differentiation remain unclear.MethodsThe expression patterns of krüppel-like transcription factors (KLFs) during megakaryocytic differentiation were identified from public databases. Phorbol 12-myristate 13-acetate (PMA) treatment of the myeloid-erythroid-leukemic cell lines K562 and HEL were used as cellular megakaryocytic differentiation models. A lentiviral transduction system was utilized to achieve the goal of amplifying or reducing KLF2. The expression of KLF2 was examined using real-time PCR and western blot. The impact of KLF2 on the megakaryocytic differentiation of K562 cells was examined by flow cytometry, Giemsa staining, Phalloidin staining and western blot. RNA-sequencing (RNA-seq) and chromatin immunoprecipitation-sequencing (ChIP-seq) technologies were used to identify the KLF2-regulated targets.ResultsKLF2 is increased in the maturation process of megakaryocytes. KLF2 overexpression accelerated the PMA-induced megakaryocytic differentiation, as reflected by an increased percentage of CD41/CD61 cells, an increased number of polyploid cells, and an elevated expression of P21 and P27. KLF2 knockdown exhibited the opposite results, indicating that KLF2 knockdown suppressed the megakaryocytic differentiation. Further, combination of the RNA-seq and ChIP-seq results suggested that chimerin 1 (CHN1) and potassium voltage-gated channel subfamily Q member 5 (KCNQ5) may be target genes regulated of KLF2. Both CHN1 and KCNQ5 knockdown could block the megakaryocytic differentiation to some content.ConclusionThis study implicated a regulatory role of KLF2 in megakaryocytic differentiation, which may suggest KLF2 as a target for illness with abnormal megakaryocytic differentiation.

Heparanase Facilitates PMA-Induced Megakaryocytic Differentiation in K562 Cells via Interleukin 6/STAT3 Pathway.

Heparanase (HPSE) is an endo-β-D-glucuronidase that cleaves heparan sulfate and hence participates in remodeling of the extracellular matrix, leading to release of cytokines that are immobilized by binding to heparan sulfate proteoglycans (HSPGs), and consequently activating signaling pathways. This function of HPSE is correlated to its expression level that is normally very low in majority of the tissues. Exceptionally, human platelets express high level of HPSE, suggesting a unique physiological role in this cell. Using K562 cell line, we found a progressive increase of HPSE during the megakaryocytic differentiation. Analysis of a series of megakaryocytic differentiation-related heparin-binding proteins (HBPs) in the cell culture medium revealed an exclusive positive correlation between the level of interleukin 6 (IL-6) and HPSE expression. IL-6 modulated megakaryocytic differentiation through activation of STAT3. Further, we demonstrated that overexpression of HPSE potentiates megakaryocytic differentiation, whereas elimination of HPSE led to a delayed differentiation. This function of HPSE is associated with its activity, as overexpression of inactive HPSE had no effect on IL-6 production and megakaryocytic differentiation. The role of HPSE is further supported by the observation in an umbilical cord blood CD34+ cells megakaryocytic differentiation model. Our data propose a novel role for HPSE in platelets production by a HPSE/IL-6/STAT3 positive feedback loop that specifically regulates megakaryocytes maturation.

ANP32A Dysregulation Contributes to Abnormal Megakaryopoiesis in Acute Megakaryoblastic Leukemia

Acute megakaryoblastic leukemia (AMKL) is characterized by hyperproliferative megakaryoblasts losing the ability to undergo terminal maturation. The mechanism underlying abnormal megakaryopoiesis in AMKL has not been fully elucidated. In this study, we discovered that ANP32A dysregulation might play a critical role. Significant upregulation of ANP32A gene was observed in online AMKL dataset and confirmed in one AMKL sample while ANP32A downregulation correlated well to human K562 and HEL cells undergoing megakaryocytic differentiation induced by PMA. These observations suggest that ANP32A may play a role in megakaryopoiesis and its dysregualtion may contribute to AMKL. However, Anp32a deficiency or overexpression only had marginal effect on normal hematopoiesis and megakaryopoiesis. In contrast, ANP32A overexpression did impair PMA-induced megakaryocytic differentiation in K562 and HEL cells with less CD61 expression and ANP32A downrgulation caused spontaneous megakaryocytic differentiation in K562 and HEL cells as well as various AMKL cells including CMK, CHRF, Meg-01, and 6133/MPL W515L evidenced by increased expression of CD41, CD61, or CD42. Mechanism study demonstrated that ANP32A knockdown promoted megakaryocyte differentiation by enhancing extracellular signal-regulated kinase (ERK) signaling to upregulate RUNX1 and FLI1 expression. Enhanced ERK phosphorylation and RUNX1 upregulation were confirmed in human primary AML cells. Furthermore, Anp32a knockdown caused apoptosis, induced cell cycle arrest at G1 phase, and dramatically impaired the ability of 6133/MPL W515L cells to form colonies in vitro. Finally, Anp32a knockdown significantly promoted mice survival in an AMKL mouse model. Mouse transplanted with 6133/MPL W515L cells (0.8 x 106 cells/mouse) died (median survival 13 days). In contrast, 5 out of 6 mice transplanted with the same amount of 6133/MPL W515L cells with ANP32A knockdown survived within 45 days (see figure). Our findings revealed a unique role of ANP32A dysregulation in retaining leukemic megakaryocytes in undifferentiated status and promoting proliferation that ultimately contributed to the pathogenesis of AMKL. Considering marginal effect of ANP32A in normal hematopoiesis and megakaryopoiesis, ANP32A may be a potential target for developing novel AMKL therapy or serve as a biomarker for diagnosis and prognosis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

ERK2-Pyruvate Kinase Axis Permits Phorbol 12-Myristate 13-Acetate-induced Megakaryocyte Differentiation in K562 Cells

Metabolic changes that contribute to differentiation are not well understood. Overwhelming evidence shows the critical role of glycolytic enzyme pyruvate kinase (PK) in directing metabolism of proliferating cells. However, its role in metabolism of differentiating cells is unclear. Here we studied the role of PK in phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic differentiation in human leukemia K562 cells. We observed that PMA treatment decreased cancer-type anabolic metabolism but increased ATP production, along with up-regulated expression of two PK isoforms (PKM2 and PKR) in an ERK2-dependent manner. Interestingly, silencing of PK (PKM2 and PKR) inhibited PMA-induced megakaryocytic differentiation, as revealed by decreased expression of megakaryocytic differentiation marker CD61 and cell cycle behavior. Further, PMA-induced ATP production reduced greatly upon PK silencing, suggesting that PK is required for ATP synthesis. In addition to metabolic effects, PMA treatment also translocated PKM2, but not PKR, into nucleus. ERK1/2 knockdowns independently and together suggested the role of ERK2 in the up-regulation of both the isoforms of PK, proposing a role of ERK2-PK isoform axis in differentiation. Collectively, our findings unravel ERK2 guided PK-dependent metabolic changes during PMA induction, which are important in megakaryocytic differentiation.

Down-regulation of superoxide dismutase 1 by PMA is involved in cell fate determination and mediated via protein kinase D2 in myeloid leukemia cells

Myeloid leukemia cells maintain a high intracellular ROS level and use redox signals for survival. The metabolism of ROS also affects cell fate, including cell death and differentiation. Superoxide dismutases (SODs) are major antioxidant enzymes that have high levels of expression in myeloid leukemia cells. However, the role of SODs in the regulation of myeloid leukemia cells' biological function is still unclear. To investigate the function of SODs in myeloid leukemia cell death and differentiation, we used myeloid leukemia cell lines K562, MEG-01, TF-1, and HEL cells for this study. We found that PMA-induced megakaryocytic differentiation in myeloid leukemia cells is accompanied by cell death and SOD1 down-regulation, while SOD2 expression is not affected. The role of SOD1 is verified when ATN-224, a SOD1 specific inhibitor, inhibits cell proliferation and promotes cell death in myeloid leukemia cells without PMA treatment. Moreover, inhibition or silencing of SODs further increases cell death and decreases polyploidization induced by PMA while they were partially reversed by SOD1 overexpression. Thus, SOD1 expression is required for myeloid leukemia cell fate determination. In addition, the knockdown of PKD2 reduces cell death and promotes polyploidization induced by PMA. PMA/PKD2-mediated necrosis via PARP cleavage involves both SOD1-dependent and -independent pathways. Finally, ATN-224 enhanced the inhibition of cell proliferation by Ara-C. Taken together, the results demonstrate that SOD1 regulates cell death and differentiation in myeloid leukemia cells. ATN-224 may be beneficial for myeloid leukemia therapy.

ZNF300 knockdown inhibits forced megakaryocytic differentiation by phorbol and erythrocytic differentiation by arabinofuranosyl cytidine in K562 cells.

Previously, we reported that ZNF300 might play a role in leukemogenesis. In this study, we further investigated the function of ZNF300 in K562 cells undergoing differentiation. We found that ZNF300 upregulation in K562 cells coincided with megakaryocytic differentiation induced by phorbol-12-myristate-13-acetate (PMA) or erythrocytic differentiation induced by cytosine arabinoside (Ara-C), respectively. To further test whether ZNF300 upregulation promoted differentiation, we knocked down ZNF300 and found that ZNF300 knockdown effectively abolished PMA-induced megakaryocytic differentiation, evidenced by decreased CD61 expression. Furthermore, Ara-C-induced erythrocytic differentiation was also suppressed in ZNF300 knockdown cells with decreased γ-globin expression and CD235a expression. These observations suggest that ZNF300 may be a critical factor controlling distinct aspects of K562 cells. Indeed, ZNF300 knockdown led to increased cell proliferation. Consistently, ZNF300 knockdown cells exhibited an increased percentage of cells at S phase accompanied by decreased percentage of cells at G0/G1 and G2/M phase. Increased cell proliferation was further supported by the increased expression of cell proliferation marker PCNA and the decreased expression of cell cycle regulator p15 and p27. In addition, MAPK/ERK signaling was significantly suppressed by ZNF300 knockdown. These findings suggest a potential mechanism by which ZNF300 knockdown may impair megakaryocytic and erythrocytic differentiation.

FMNL1 Is Downregulated In Myelodysplastic Syndromes and Is Involved In Megakaryocytic Differentiation

BackgroundMyelodysplastic syndromes (MDS) are disorders characterized by morphological dysplasia, impaired differentiation and defective cellular functions, resulting in peripheral cytopenias. FMNL1 belongs to a family of formin-related proteins, indispensable for many fundamental actin-dependent processes. Recently, FMNL1 has been described to be upregulated and play a role in the actin cytoskeleton dynamics during monocyte differentiation to macrophages. AimsThe aim of this work was to characterize FMNL1 expression in total bone marrow cells of patients with MDS comparing to normal donors. We also analyzed FMNL1 expression in erythrocytic, granulocytic and megakaryocytic differentiation, using cell line models. Finally, we evaluated the impact of inhibition of FMNL1 during megakaryocytic differentiation. MethodsA total of 49 patients with a diagnosis of MDS, receiving no treatment, and 18 samples from normal donors were included in the study, which was approved by the National Ethical Committee Board. Samples were submitted to RNA extraction after removal of erythrocytes by hemolysis. FMNL1 expression levels from cell lines or total bone marrow cells were determined by quantitative PCR (q-PCR) or Western blot. KU812 was treated 50 μM hemin and 100 μM hydroxyurea for erythrocytic differentiation. K562 was stimulated with 20nM of PMA for megakaryocytic differentiation. NB4 was treated with 10-6 M of ATRA for granulocytic differentiation. Megakaryocytic differentiation was followed by the increase in megakaryocytic marker (CD61) determined by flow cytometry and cells were also stained with May–Grunwald–Giemsa. K562 cells were transduced with lentivirus-mediated shRNA targeting LacZ or FMNL1. Apoptosis was assessed by Annexin-V/PI staining and cell cycle was evaluated by flow cytometry, both at 24 and 48 hours after induction with PHA. The statistical methods used were the age-adjusted multivariate linear regression analysis, Mann Whitney test or t test. ResultsFMNL1 expression in bone marrow samples was significantly lower in MDS when compared with normal donor cells (P=0.01), especially in the high risk group (P<0.02). Using cell line models for hematopoietic differentiation, there was a fifteen-fold increase and a five-fold increase in FMNL1 expression for megakaryocytic (P=0.002) and granulocytic differentiation (P=0.05) respectively. Western blot analysis corroborated these findings. There was no difference for erythrocytic differentiation. After PMA treatment, the level of the megakaryocytic markers CD61 was significantly lower in K562 shFMNL1 when compared with shLacZ (P=0.01). The level of CD41a (P=0.5) and CD42b (P=0.1) showed a trend toward a decrease in K562 shFMNL1 when compared with shLacZ, however not statistically significantly. Corroborating these data, K562 shFMNL1 showed abnormal megakaryocytic morphological features (larger cells with polylobulated or polysegmented nuclei and vacuolization) compared with cells shLacZ. There were no statistical differences in the apoptosis levels and cell cycle analysis between K562 shFMNL1 and shLacz cells. ConclusionsThe family of formin-related proteins has mainly been related to actin-dependent processes although little is known regarding their possible involvement in haematopoiesis. The lower FMNL1 expression in MDS BM could reflect the role of this protein in cell differentiation. We were therefore prompted to study this issue in depth using megakaryocytic differentiation as a system. In this study, we show increased expression of FMNL1 in PMA-induced megakaryocytic differentiation of K562 cells. Furthermore, knockdown of FMNL1 deregulates differentiation, suggesting that FMNL1 is required in order to maintain the effective megakaryocytopoiesis in MDS. Although FMNL1 silencing effectively down-regulated CD61 expression, CD41a and CD42b were reduced to a lesser extent. The effect of FMNL1 cannot be explained by modifications of the cell cycle or apoptosis during differentiation; and is probably due to the effect in changes in the dynamic remodeling of the cytoskeleton. Disclosures:No relevant conflicts of interest to declare.

Megakaryocytic differentiation in human chronic myelogenous leukemia K562 cells induced by ionizing radiation in combination with phorbol 12-myristate 13-acetate

Differentiation-induction therapy is an attractive approach in leukemia treatment. It has been suggested that the accumulation of intracellular reactive oxygen species (ROS) is involved in megakaryocytic differentiation induced by phorbol 12-myristate 13-acetate (PMA) in the K562 leukemia cell line. Therefore, a ROS-inducible technique could be a powerful method of differentiation induction. Accordingly, we hypothesized that ionizing radiation contributes to the acceleration of megakaryocytic differentiation through the accumulation of intracellular ROS in leukemia cells. In the present study, ionizing radiation was shown to promote PMA-induced megakaryocytic differentiation. Cells with high CD41 expression sustained intracellular ROS levels effectively. The enhancement of differentiation by ionizing radiation was found to be regulated through the mitogen-activated protein kinase (MAPK) pathway, involving both extracellular signal-regulated protein kinase 1/2 (ERK1/2) and p38 MAPK. Ionizing radiation also controlled mRNA expression of the oxidative stress response gene heme oxygenase-1 (HO1). Consequently, we concluded that intracellular ROS, increased by ionizing radiation, modulate megakaryocytic differentiation downstream of the MAPK pathway.

PHD Domain Deletion Mutations of ASXL1 Promote Myeloid Leukemia Transformation Through Epigenetic Dysregulation and Inhibit Megakaryocytic Differentiation Through the Inactivation of FOSB in K562 Cells.

AbstractAbstract 2393Somatic mutations of ASXL1 gene have been described in patients with myeloid malignancies and were associated with inferior outcomes. ASXL1 mutations have also been detected in myeloid blast crisis of chronic myeloid leukemia (CML) patients. The mechanisms of acute myeloid leukemia (AML) transformation and functional role of ASXL1 mutations in the leukemogenesis remain to be determined. Recently, we identified PHD domain deletion mutations (R693X and L885X) in patients with CML in myeloid blast crisis and/or AML with minimal differentiation (M0). In the present study, we aimed to investigate the role of PHD domain deletion mutations in the pathogenesis of AML transformation. The K562 cells carrying Philadelphia chromosome, serves as a model to study the molecular mechanisms associated with leukemogenesis. Our result showed that R693X/L885X mutations inhibited PMA-treated megakaryocytic differentiation with the change of physiological characteristic features and suppressed the induction of CD61, a specific cell surface marker of megakaryocytes. We also found that FOSB, a member of Fos family of AP-1 transcription factors was down-regulated in K562 cells expressing R693X and L885X compared to wild-type ASXL1 during PMA-mediated megakaryocytic differentiation. Examination of intracellular signaling pathways showed that the mutant ASXL1 protein prevented PMA-induced megakaryocytic differentiation through the inactivation of ERK, AKT and STAT5 which are required for differentiation. Further, ASXL1 depletion by shRNA in K562 cells led to enhanced cell proliferation, increased colony formation and impaired PMA-mediated differentiation. Previous studies in Drosophila had revealed that Asxl forms the protein complexes of both Trithorax and Polycomb groups that are required for maintaining chromatin in both activated and repressed transcriptional states. By using Western blot analysis, we demonstrated that PHD domain deletion mutations of ASXL1 significantly suppressed the transcriptionally repressive mark H3K27 trimethylation, however no effect on methylated H3K4 (H3K4me2 and H3K4me3), an active histone mark in K562 cells. Co-immunoprecipitation analysis revealed that wild-type, but not PHD domain deletion mutations of ASXL1 interact with EZH2, a member of the polycomb repressive complex 2 (PRC2). Importantly, PHD deletion mutations or downregulation of ASXL1 resulted in the suppression of EZH2 in K562 cells. Our study demonstrated that PHD deletion mutations of ASXL1 resulted in a loss-of-function which exhibited direct effects on the proliferation and differentiation and also proposed a specific role for ASXL1 in epigenetic regulation of gene expression in K562 cells. Disclosures:No relevant conflicts of interest to declare.

Small Rab GTPase Rab7b Promotes Megakaryocytic Differentiation by Enhancing IL-6 Production and STAT3-GATA-1 Association.

AbstractAbstract 1549Interleukin 6 (IL-6) is a pleiotropic cytokine acting on a variety of cell types, and plays important roles in hematopoiesis. GATA-1 (GATA binding protein 1) is an important transcription factor involved in either megakaryocytic or erythrocytic differentiation. However, the mechanisms for IL-6-induced megakaryocytic differentiation and regulation of GATA-1 have not been fully elucidated. By using phorbol-12-myristate-13-acetate (PMA)-induced megakaryocytic differentiation of K562 cells as a model, we investigated the roles of Rab7b, a late endosome/lysosome-localized myeloid small GTPase, in megakaryocytic differentiation. We find that Rab7b can promote PMA-induced megakaryocytic differentiation, as evidenced by typical morphological alterations, increased fibronectin-specific adhesion, increased polyploidy formation, and increased expression of CD41a. The GTP-bound status and lysosomal localization of Rab7b are required for promotion of K562 megakaryocytic differentiation, which can be blocked by inhibitor of nuclear factor κB (NF-κB) and neutralizing antibodies for IL-6 and gp130. In Rab7b-silenced K562 cells, PMA-induced activation of NF-κB, IL-6 production and megakaryocytic differentiation are impaired. Furthermore, we demonstrate that IL-6-induced activation of signal transducer and activator of transcription 3 (STAT3) and the subsequent association of STAT3 with GATA-1 may contribute to PMA-induced and Rab7b-mediated transcriptional upregulation of megakaryocytic differentiation markers. Therefore, our data suggest that Rab7b may play important roles in megakaryopoiesis by activating NF-κB and promoting IL-6 production. Our study also indicates that the IL-6-induced association of STAT3 with GATA-1 may favor megakaryopoiesis. Disclosures:No relevant conflicts of interest to declare.

Small Rab GTPase Rab7b promotes megakaryocytic differentiation by enhancing IL-6 production and STAT3-GATA-1 association

Induction of the differentiation of human leukemia cells is a useful strategy in treatment of human leukemia. However, the molecular mechanisms involved in leukemia cell differentiation have not been fully elucidated. Interleukin 6 (IL-6) is a pleiotropic cytokine acting on a variety of cell types, and plays important roles in hematopoiesis. GATA binding protein 1 (GATA-1) is an important transcription factor involved in either megakaryocytic or erythrocytic differentiation. Herein we report that Rab7b, a late endosome/lysosome-localized myeloid small GTPase, promotes phorbol-12-myristate-13-acetate (PMA)-induced megakaryocytic differentiation by increasing nuclear factor κB (NF-κB)-dependent IL-6 production and subsequently enhancing the association of activated signal transducer and activator of transcription 3 (STAT3) with GATA-1. By using PMA-induced megakaryocytic differentiation of leukemia cells as a model, we investigated the roles of Rab7b in megakaryocytic differentiation. We find that Rab7b can potentiate PMA-induced upregulation of megakaryocytic markers, production of IL-6, and activation of NF-κB. Inhibitor of NF-κB and neutralizing antibodies for IL-6 or the IL-6 signaling receptor gp130 can block the effects of Rab7b in megakaryocytic differentiation. In Rab7b-silenced cells, PMA-induced activation of NF-κB, IL-6 production, and megakaryocytic differentiation are impaired. Furthermore, we demonstrate that IL-6-induced activation of STAT3 and the subsequent association of STAT3 with GATA-1 may contribute to PMA-induced and Rab7b-mediated transcriptional upregulation of megakaryocytic differentiation markers. Therefore, our data suggest that Rab7b may play important roles in megakaryopoiesis by activating NF-κB and promoting IL-6 production. Our study also indicates that the IL-6-induced association of STAT3 with GATA-1 may regulate megakaryocytic differentiation.

Protein-arginine Methyltransferase 1 Suppresses Megakaryocytic Differentiation via Modulation of the p38 MAPK Pathway in K562 Cells

Protein-arginine methyltransferase 1 (PRMT1) plays pivotal roles in various cellular processes. However, its role in megakaryocytic differentiation has yet to be investigated. Human leukemia K562 cells have been used as a model to study hematopoietic differentiation. In this study, we report that ectopic expression of HA-PRMT1 in K562 cells suppressed phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic differentiation as demonstrated by changes in cytological characteristics, adhesive properties, and CD41 expression, whereas knockdown of PRMT1 by small interference RNA promoted differentiation. Impairment of the methyltransferase activity of PRMT1 diminished the suppressive effect. These results provide evidence for a novel role of PRMT1 in negative regulation of megakaryocytic differentiation. Activation of ERK MAPK has been shown to be essential for megakaryocytic differentiation, although the role of p38 MAPK is still poorly understood. We show that knockdown of p38alpha MAPK or treatment with the p38 inhibitor SB203580 significantly enhanced PMA-induced megakaryocytic differentiation. Further investigation revealed that PRMT1 promotes activation of p38 MAPK without inhibiting activation of ERK MAPK. In p38alpha knockdown cells, PRMT1 could no longer suppress differentiation. In contrast, enforced expression of p38alpha MAPK suppressed PMA-induced megakaryocytic differentiation of parental K562 as well as PRMT1-knockdown cells. We propose modulation of the p38 MAPK pathway by PRMT1 as a novel mechanism regulating megakaryocytic differentiation. This study thus provides a new perspective on the promotion of megakaryopoiesis.

Rab7b Promotes Megakaryocytic Differentiation of K562 Cells by Activating the Protein Kinase C/Extracellular Signal-Regulated Kinase Dependent Pathway.

Abstract 3612Poster Board III-548Rab proteins are small GTPases belonging to the Ras superfamily and involved in cell protein transportation processes such as internalization and exocytosis. Recently it has been found that several Rab proteins such as Rab7 are also essential for signaling and the controlling of cell proliferation and differentiation. We previously have identified a small GTPase homologous to Rab7, named Rab7b, which is localized to lysosome-associated compartments and is selectively expressed in promyeloid or monocytic cells. We demonstrated that rab7b is involved in monocytic differentiation of human acute promyelocytic leukemia cells. In this study, we detected the expression level of Rab7b during the process of PMA induced megakaryocytic differentiation of K562 cells, transfected Rab7b and its mutants into K562 cells, and investigated the function and signaling mechanism of PMA induced megakaryocytic differentiation of K562 cells. Firstly, we treated K562 cells with PMA for various times, detected the expression level of Rab7b by quantitative PCR and Western blot, and measured the expression level of CD11b, CD14, CD33 and CD41a on the cell surface. Next, we constructed Rab7b, Rab7b-Q67L (active GTPase-deficient mutant), Rab7b-ΔCC (localization deficiency) and Rab7b-RNAi expression vectors, which were then transfected into K562 cells. Megakaryocytic differentiation of cells were analyzed after treated with PMA for various times, including cell morphology and adhesive properties, cell growth arrest, polyploidization, secretion of IL-6 and expression of specific marker CD41a. Because of the considerable role of PKC and ERK1/2 signaling pathways in the megakaryocytic differentiation of K562 cells, we treated various K562 cells with PMA for various times and detected the activation of ERK1/2 by Western blot. Later, cells were inhibited by PKC and ERK1/2 specific inhibitors and treated with PMA for various times, and then the expression of Rab7b and megakaryocytic differentiation of K562 cells were observed. At last, PMA-induced activation of PKC and ERK1/2 signaling has been suggested to regulate cell cycle arrest and megakaryocyte differentiation by modulating the expression of the p21waf/cip1 and the dephosphorylation of the Rb protein, which were then detected by us after transfection of Rab7b and inhibition of PKC/MAPK pathways in K562 cells. The expression of Rab7b was upregulated during the PMA-induced megakaryocytic differentiation of K562 cells. Overexpression of wild-type and active mutant (Q67L) of Rab7b enhanced PMA stimulated megakaryocytic differentiation and ERK1/2 activation. In addition, overexpression of the localization deficient mutant (ΔCC) or RNAi of Rab7b inhibited PMA-stimulated megakaryocytic differentiation and ERK1/2 activation. PMA-induced megakaryocytic differentiation and activation of ERK1/2 in K562 cells was reversed by inhibiting either PKC or ERK1/2 signaling pathway, whereas the mRNA and protein expression of Rab7b was only suppressed in PKC inhibiting groups, indicating that Rab7b mediates a signal for megakaryocytic differentiation downstream of PKC and upstream of ERK1/2. Therefore, we concluded that Rab7b could promote PMA induced differentiation of K562 cells by activating the PKC/ERK1/2 signaling pathway and provide Rab7b as a candidate to connect the PKC pathway and the ERK1/2 pathway in PMA-induced megakaryocytic differentiation of K562 cells. Disclosures:No relevant conflicts of interest to declare.

Early growth response transcription factor EGR-1 regulates Galphaq gene in megakaryocytic cells.

Galphaq (Gene GNAQ) plays a major role in platelet signal transduction but little is known regarding its transcriptional regulation. We studied Galphaq promoter activity using luciferase reporter gene assays in human erythroleukemia (HEL) cells treated with phorbol 12-myristate 13-acetate (PMA) for 24 h to induce megakaryocytic transformation. PMA-treated HEL cells showed enhanced Galphaq expression. Reporter (luciferase) gene studies on 5' upstream construct (up to -116 bp from ATG) revealed a negative regulatory site at -238/-202 and two positive sites at -203/-138 and -1116/-731. The positive regulatory region -203/-138 contained overlapping Sp1/AP-2/EGR-1 consensus sites. Gel shift studies on Galphaq oligonucleotides 1 (-203/-175) and 2 (-174/-152) using HEL cell extracts demonstrated protein binding that was due to early growth response factor EGR-1 at two sites. Mutations in either EGR-1 site markedly decreased the gene activity, indicating functional relevance. Mutation of consensus E-Box motif (-185/-180) had no effect. Reduction in the expression of endogenous EGR-1 with antisense oligonucleotide to EGR-1 inhibited PMA-induced Galphaq transcription. Correspondingly, Egr-1 deficient mouse platelets also showed approximately 50% reduction in the Galphaq expression relative to wild-type platelets. These studies suggest that Galphaq gene is regulated during PMA-induced megakaryocytic differentiation by EGR-1, an early growth response transcription factor that regulates a wide array of genes and plays a major role in diverse activities, including cell proliferation, differentiation and apoptosis, and in vascular response to injury and atherosclerosis.

Differential expression changes in K562 cells during the hemin-induced erythroid differentiation and the phorbol myristate acetate (PMA)-induced megakaryocytic differentiation

K562 cell line has been used as a model of common progenitor of erythroblasts and magakaryocytes and can be differentiated into erythroid and megakaryocytic lineages by hemin and phorbol myristate acetate (PMA) respectively. We analyzed mRNA expression in un-induced, hemin-induced and PMA-induced K562 cells by differential display reverse transcription polymerase chain reaction (DDRT-PCR) method. 314 differential expression sequence tags (ESTs) were obtained. Among them, 201 ESTs displayed up-regulation and 85 ESTs down-regulation after hemin induction, 186 ESTs showed up-regulation and 72 ESTs down-regulation after PMA induction. The differentially expressed genes included those encoding transcription factors, signaling factors, apoptosis-associated factors and others. 45 of these ESTs stand for genes whose open reading frames were found but whose functions remain unknown. 4 ESTs represent possibly new genes. Furthermore we compared differences of gene expression during hemin-induced erythroid differentiation and PMA-induced megakaryocytic differentiation and found that the expressional changes of some transcription factors and metabolism proteins are the common but the expressional changes of some signal pathways in these two differentiation processes are different. These results suggested that erythroid differentiation and megakaryocytic differentiation are associated in activation and repression of different signal pathways.

Identification and characterization of a novel zinc finger protein (HZF1) gene and its function in erythroid and megakaryocytic differentiation of K562 cells

A novel zinc finger protein (HZF1) gene was identified and characterized by screening a human bone marrow cDNA library, using a new expression sequence tag probe that contains sequences encoding zinc finger motifs. There are at least three transcripts that may result from different splicing of the pre-mRNA, but the differences among them are only involved in 5' non-translation region of HZF1 mRNA. HZF1 gene contains four exons and three introns. The putative protein consists of 670 amino-acid residues including 15 typical C2H2 and 2 C2RH zinc finger motifs. This structure characterization of HZF1 and the nuclear location of the protein suggest that HZF1 may function as a transcription factor. HZF1 mRNA expression was detected in ubiquitous tissues and various hematopoietic cell lines. Increased HZF1 mRNA expression was observed following erythroid differentiation of K562 cells induced by hemin or megakaryocytic differentiation of K562 cells induced by phorbol myristate acetate (PMA). Both of the antisense method and RNA interference assay revealed that repression of the intrinsic expression of HZF1 blocked the hemin-induced erythroid differentiation and reduced the PMA-induced megakaryocytic differentiation of K562 cells, which suggested that HZF1 play important roles in erythroid and megakaryocytic differentiation.

Identification, Characterization of a Novel Zinc Finger Protein (HZF1) Gene and Its Roles in Erythroid Differentiation and Megakaryocyte Differentiation.

A significant number of transcription factors contain evolutionarily conserved zinc finger motifs. The classical C2H2 zinc finger motif, which employs two cysteine and two histidine residues to coordinate a single zinc ion, is a maim type of the zinc finger proteins. Many of the identified C2H2 type zinc protein have been demonstrated to be transcription factors that play important roles in differentiation and development of cells and tissues of higher organisms. In this study, we obtained some novel expression sequence tags (ESTs) containing C2H2 type motifs by reverse transcription-polymerase chain reaction (RT-PCR) using RNAs derived from hemin-induced K562 cells. A cDNA encoding novel zinc finger protein (designed as HZF1) was obtained by screening the human bone marrow cDNA library using one of the ESTs as the probe. The cDNA sequences (2013 nucleotides) have been submitted to the GenBank databases under accession No. AF244088.1). Three transcripts of HZF1 gene were explored by PCR amplification of cDNAs derived from hemin-induced K562 cells. The cDNA sequences (2632 nucleotides) of the longest transcript have been submitted to the GenBank databases under accession No. DQ117529). These transcripts may result from different splicing of the pre-mRNA of HZF1 but the differences between them are only involved in 5′ non-translation region of HZF1 mRNA. BLASTN analysis revealed that HZF1 gene has four exons and three introns. The putative protein consists of 670 amino acid residues including continuous 15 C2H2 and 2 C2RH zinc finger motifs. This structure characterization and the nuclear location of the protein suggest that HZF1 may function as a transcription factor. HZF1 mRNA was detected in ubiquitous tissues and various hematopoietic cell lines. Increased HZF1 mRNA expression was observed following hemin-induction or phorbol myristate acetate (PMA)-induction of K562 cells. Both of the antisence method and RNA interference assay revealed that repression of the intrinsic expression of HZF1 blocked the hemin-induced erythroid differentiation and reduced the PMA-induced megakaryocytic differentiation of K562 cells, which suggested that HZF1 play an important part in erythroid differentiation and megakaryocytic differentiation.

Oncogenic potential of the transcription factor LYL1 in acute myeloblastic leukemia

The LYL1 gene encodes a basic helix-loop-helix transcription factor involved in T-cell acute lymphoblastic leukemia. Using real-time quantitative RT-PCR assay, we found that the expression of LYL1 was at higher levels in the majority cases of acute myeloblastic leukemia (AML) or myelodysplastic syndrome when compared to normal bone marrow. Our study also showed that LYL1 was highly expressed in most AML cell lines and in CD34+ AML cells. To determine whether LYL1 had an affect on the phenotype and behavior of myeloid cells, we introduced full-length LYL1 cDNA into K562 cells using electroporation and U937 cells with retroviral infection. Both of the derivative cell lines with overexpression of LYL1 had an increased growth rate and clonogenecity. Forced expression of LYL1 in K562 cells enhanced spontaneous and hemin-induced erythroid differentiation but blocked spontaneous as well as PMA-induced megakaryocytic differentiation. Overexpression of LYL1 in U937 cells blocked all-trans retinoic acid-induced monocytic differentiation. The LYL1-transfected U937 cells were also more resistant to the cytotoxic drug cytarabine. These results demonstrate that LYL1 may play a role in early hematopoiesis and may be a potential oncogenic factor in AML.

Physical and Functional Interactions between Mitotic Kinases during Polyploidization and Megakaryocytic Differentiation

Human Polo-like kinase 3 (Plk3), a protein serine/threonine kinase, is involved in the regulation of cell cycle progression at multiple stages. Our previous studies revealed that Plk3 is closely associated with centrosomes and plays an important role in the regulation of microtubule dynamics. Here we describe the physical interaction of Plk3 with Aurora A and BubR1 kinases, and the significance of this interaction during terminal differentiation and polyploidization of megakaryocytes. Specifically, double immunofluorescence staining confirms that Plk3 and Aurora A co-localize to centrosomes or spindle poles during essentially all phases of the cell cycle and that BubR1 also exhibits spindle pole localization during metaphase. Pull-down assays show that Plk3 physically interacts with Aurora A as well as BubR1. Upon treatment with phorbol 12-myristate 13-acetate (PMA), human erythroleukemic cells (K562) underwent megakaryocytic differentiation characterized by polyploidization and expression of mature megakaryocyte surface markers such as CD41. Plk3 protein levels were seen to be increased during PMA-induced megakaryocytic differentiation of K562 cells, correlating well with the ploidy level in these cells. Similarly, Aurora A and its phosphorylated form also increased after PMA treatment. In contrast, BubR1 levels were markedly reduced. Taken together, our study suggests that Plk3 and Aurora A kinases may lie in the same regulatory pathway and that Plk3 and Aurora A as well as BubR1 may play an important role in polyploidization and megakaryocytic differentiation.

The protein tyrosine phosphatase HePTP regulates nuclear translocation of ERK2 and can modulate megakaryocytic differentiation of K562 cells.

In response to PMA treatment K562 myelogenous leukemia cells undergo megakaryocytic differentiation, which is dependent on prolonged ERK activation and is characterized by growth arrest, upregulation of CD41 and IL-6, and, finally, by characteristic changes in cell morphology. The tyrosine phosphatase HePTP was recently demonstrated to regulate ERK activity and changes in HePTP expression have been associated with hematopoietic malignancies. Here, we have studied the function of HePTP during PMA-induced megakaryocytic differentiation of K562 cells. Overexpression of HePTP or inhibition of HePTP expression with antisense cDNA had no effect on PMA-induced cell cycle arrest or upregulation of cyclin D in K562 cells. The expression of megakaryocytic markers such as CD41 and IL6, however, were highly reduced in cells overexpressing HePTP, due to reduced ERK activation, and the cells were impaired in their ability to differentiate. Compared to control cells, HePTP antisense expressing cells did not show increased basal or PMA-induced ERK activity. However, antisense inhibition of HePTP enhanced nuclear translocation of ERK and the expression of the megakaryocytic markers CD41 and IL-6. Interestingly, like cells overexpressing HePTP, morphological differentiation was also impaired in HePTP antisense expressing cells. The results for the first time demonstrate that different aspects of megakaryocytic differentiation have distinct requirements for ERK activity. They further show that HePTP is involved in the regulation of nuclear translocation of ERK2 and that HePTP protein levels can modulate K562 cell differentiation.