Megakaryocytic Differentiation Of K562 Cells Research Articles

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.

Stromal Contact Inhibits Effects of Phorbol Esters but Does Not Inhibit Staurosporine Induced Megakaryocytic Differentiation in K562 Cells.

Development of human megakaryocytes is modulated at least in part by direct contact with stromal cells, however, the mechanisms that control this process have not been fully elucidated. Stromal contact is not needed for lineage commitment and direct contact of human progenitor cells with marrow stroma negatively influences megakaryocytic development. The K562 human erythroleukemia cell line has been extensively used as a model to study molecular aspects of megakaryocytic differentiation. Treatment of K562 with either phorbol-12 myristate-13 acetate (PMA), a PKC activator, or staurosporine (STSP) a PKC inhibitor, induces K562 cells to undergo differentiation to a megakaryocytic phenotype. Treated cells undergo growth arrest, changes in cellular morphology as well as increased expression of megakaryocytic/platelet-specific antigens, such as CD61. We have demonstrated that PMA-induced differentiation of K562 cells is inhibited by co-culture on the bone marrow stromal cell line, OP9. These cells remained small, continued to proliferate, and had less than 10% CD61 surface expression, similar to untreated K562. In contrast, K562 cells co-cultured on OP9 cells and treated with STSP retained the ability to differentiate. They were significantly larger than unstimulated K562 cells, exhibited megakaryocytic morphology, and had increased CD61 surface expression from 3.5±2.4% at baseline to 81±2.4%. These data suggest that while STSP and PMA recapitulate certain aspects of megakaryocytic differentiation, divergent signaling pathways may regulate the effects of stromal inhibition. Since stromal inhibition of differentiation plays an important role in the maintenance of hematopoietic progenitors in normal marrow, we investigated the molecular mechanisms involved in STSP induced differentiation. PMA induced megakaryocytic differentiation is dependent on sustained activation of ERK/MAPK, and stromal inhibition is associated with a loss of sustained Rap1 activation. The mechanism of action for STSP induced megakaryocytic differentiation has not been studied. Therefore we sought to investigate the ERK/MAPK signaling pathway during STSP induced differentiation. Using western blot analysis we found the small G protein c-RAF was phosphorylated at Ser338 when K562 cells were treated with either PMA or STSP, suggesting both agents activate c-RAF. In turn both led to a dramatic increase in ERK and p90RSK phosphorylation. We next examined the ERK signaling cascade during stromal inhibition. K562 cells co-cultured on OP9 cells and treated with PMA showed only a slight increase in c-RAF phosphorylation and no significant ERK or p90RSK phosphorylation, consistent with previous stromal inhibition data. However, STSP treatment of K562 co-cultured cells led to the robust phosphorylation of c-RAF and ERK. P90RSK was not significantly phosphorylated. Taken together these data suggest STSP treatment leads to activation of the ERK/MAPK pathway and that this activation may be Rap1 independent. Understanding the mechanisms involved in stromal inhibition and the divergent signaling pathways involved in megakaryocytic differentiation may bring to light novel functions of marrow stroma during normal and leukemic cell expansion.

The HIV-1 Tat Protein Selectively Enhances CXCR4 and Inhibits CCR5 Expression in Megakaryocytic K562 Cells

The hematopoietic compartments act as long-term reservoirs for human immunodeficiency virus type-1 (HIV-1). Although hematopoietic progenitor cells (HPCs) are rarely infectable, HPCs committed to the megakaryocytic lineage can be infected and support a productive infection by both the X4 and R5 strains of HIV-1. Indeed, in contrast to the CD34+ progenitors, the lineage-committed HPCs express high levels of the HIV-1 co-receptors, CXCR4 and CCR5. The HIV-1 transactivator (Tat) protein has been shown to alter co-receptor expression in T lymphocytes and macrophages. We hypothesized that Tat may regulate co-receptor expression in lineage-specific HPCs as well. We have monitored the effects of Tat protein on co-receptor expression and on lineage-specific differentiation, using the HPC cell line, K562. Butyric acid (BA)-induced erythroid differentiation in K562 cells was suppressed by 1-100 ng/ml of Tat, as evident from a 70-80% decrease in hemoglobin (Hb) production and a 10-30-fold decrease in glycophorin-A expression. However, Tat treatment enhanced phorbol myristate acetate (PMA)-induced megakaryocytic differentiation, as evident from a 180-210% increase in 3H-serotonin uptake and a 5-12-fold increase in CD61 expression. Tat did not significantly alter co-receptor expression in erythroid cells. However, Tat co-treatment profoundly effected both CXCR4 and CCR5 gene expression and protein levels in megakaryocytic cells. In PMA-stimulated cells, Tat increased CXCR4 and decreased in CCR5 expression, this was potentiated in cells chronically exposed to Tat. In conclusion, Tat protein suppresses erythroid and facilitates megakaryocytic differentiation of K562 cells. In megakaryocytic cells, Tat differentially effected CXCR4 and CCR5 expression. Because megakaryocytes may play a crucial role in HIV-1 infectivity in viral reservoirs, our findings implicate a role for Tat protein in dictating co-receptor usage in lineage-committed HPCs.

Humanin delays apoptosis in K562 cells by downregulation of P38 MAP kinase

Humanin (HN) is a newly identified neuroprotective peptide. In this study, we investigated its antiapoptotic effect and the potential mechanisms in K562 cells. Upon serum deprivation, expression of HN in K562 cells decreased and its intracellular distribution changed from cytoplasm to cell membrane. In HN stably transfected K562 cells, apoptosis was delayed compared with control vector transfected cells as measured by flow cytometry. Furthermore, analysis of different mitogen-activated protein (MAP) kinases activity revealed that extracellular signal-regulated kinase (ERK) pathway was inhibited while p38 signaling was activated following serum deprivation in K562 cells. And in HN transfected K562 cells, ERK downregulation was not affected, but p38 activation was suppressed, which may responsible for the delayed apoptosis in these cells. Activation of the ERK signaling pathway by phorbol myristate 13-acetate (PMA) and sorbitol protected K562 cells from serum deprivation induced apoptosis. Additionally, overexpression of HN reduced megakaryocytic differentiation of K562 cells. The present data outline the role of ERK and p38 MAP kinases in serum deprivation induced apoptosis in K562 cells and figure out p38 signaling pathway as molecular target for HN delaying apoptosis in K562 cells.

Autocrine signaling of platelet-derived growth factor regulates disabled-2 expression during megakaryocytic differentiation of K562 cells

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 STI571-treated 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.

Decursin and PDBu: Two PKC activators distinctively acting in the megakaryocytic differentiation of K562 human erythroleukemia cells

Protein kinase C (PKC) plays an important role in the proliferation and differentiation of various cell types including normal and leukemic hematopoietic cells. Phorbol 12,13-dibutyrate (PDBu) induces the megakaryocytic differentiation of K562 human erythroleukemia cells through PKC activation. Decursin, a pyranocoumarin from Angelica gigas, exhibits the cytotoxic effects on various human cancer cell lines and in vitro PKC activation. We report here the differences between two PKC activators, tumor-suppressing decursin and tumor-promoting PDBu, in their actions on the megakaryocytic differentiation of K562 cells. First of all, decursin inhibited PDBu-induced bleb formation in K562 cells. Decursin also inhibited the PDBu-induced megakaryocytic differentiation of K562 cells that is characterized by an increase in substrate adhesion, the secretion of granulocyte/macrophage colony stimulating factor (GM-CSF) and interleukin-6 (IL-6), and the surface expression of integrin β3. The binding of PDBu to PKC was competitively inhibited by decursin. Decursin induced the more rapid down-regulation of PKC α and βII isozymes than that induced by PDBu in K562 cells. Unlike PDBu, decursin promoted the translocation of PKC α and βII to the nuclear membrane. Decursin-induced faster down-regulation and nuclear translocation of PKC α and βII were not affected by the presence of PDBu. All these results indicate that decursin and phorbol ester are PKC activators distinctively acting in megakaryocytic differentiation and PKC modulation in K562 leukemia cells.

AP-1 regulates α 2β 1 integrin expression by ERK-dependent signals during megakaryocytic differentiation of K562 cells

Mitogen-activated protein kinases (MAPKs) have been implicated as regulators of cellular differentiation. The biological effect of MAPK signaling in the nucleus is achieved by signal-responsive transcription factors. Here, we have investigated the connection of MAPKs, transcription factor AP-1, and α 2β 1 integrin expression in K562 cells undergoing differentiation along the megakaryocytic pathway. We report that three distinct MAPKs, ERK, JNK, and p38, are activated during the TPA-induced megakaryocytic differentiation. Activation of MAPK pathways is followed by acquisition of the AP-1 DNA-binding and transactivation capacities. AP-1 DNA-binding activity consists primarily of JunD, c-Fos, and Fra-1, and is accompanied with the increased expression and phosphorylation of these subunits. While inhibition of JNK mainly prevents expression and phosphorylation of JunD and c-Jun, inhibition of the ERK pathway suppresses both phosphorylation and expression of Jun proteins, and expression of c-Fos and Fra-1. Furthermore, only the activity of the ERK pathway is essential for the differentiation response, as determined by expression of α 2β 1 (CD49b) integrin. The importance of AP-1 as a mediator ERK signaling during differentiation is demonstrated by the findings that expression of c-fos siRNA and dominant negative AP-1/c-Jun bZIP downregulate the TPA- and ERK-induced expression of α 2β 1 integrin mRNAs and proteins. Conversely, coexpression of JunD or c-Jun and c-Fos can induce α 2β 1 integrin expression independently of upstream signals. Taken together, the results show that AP-1 is a nuclear target of the ERK-pathway and mediates α 2β 1 integrin expression during megakaryocytic differentiation of K562 cells.

Identification of Signaling-Induced Transcription Pathway Determinants that Influence Erythro-Megakaryocytic Lineage Commitment.

Erythroid and megakaryocytic cells are derived from a common precursor and share many of the same transcriptional regulators. We and others have demonstrated a critical role for sustained activation of extracellular-related (ERK) kinase in PKC-induced megakaryocytic differentiation of K562 cells. However, the transcriptional mechanisms underlying this process are largely unknown. Our prior studies have focused on late stage megakaryocyte specific genes such as glycoprotein IIb/CD41. In order to understand the early transcriptional events regulating this process, we have begun to examine the mechanisms regulating the expression of CD9, a tetraspanin molecule that is currently the earliest identified marker of megakaryocyte differentiation. The PKC agonist ingenol 3,20 dibenzoate (IDB) rapidly induced CD9 expression in both K562 cells as well as in primary human CD34+ progenitor cells, much earlier than the kinetics for CD41 induction. CD9 induction by PKC was dependent on ERK activation. Recently, it has been shown that immediate early gene expression, including the transcription factor Egr-1, is highly sensitive to the duration and amplitude of ERK activity. Egr-1 has been previously implicated in the regulation of hematopoietic lineage commitment. PKC/ERK activation rapidly and strongly induced Egr-1 expression, which remained elevated for up to 96 hrs after IDB treatment of K562 cells. Egr-1 induction was closely followed by CD9 induction, suggesting a causal relationship. This was supported by chromatin immunoprecipitation (ChIP) analysis, which showed strong recruitment of Egr-1 to the CD9 promoter during PKC/ERK-induced differentiation. KLF5, a known downstream target of Egr-1, was identified as an additional target of PKC/ERK signaling during megakaryocytic differentiation. KLF5, a novel GC binding factor, was found to be expressed in primary megakaryocytes and not in erythroid cells. KLF5 expression was induced by IDB treatment, and ChIP analysis showed that Egr-1 was recruited to the KLF5 promoter during PKC/ERK-induced differentiation. KLF5 has been shown previously to regulate known megakaryocyte-expressed genes PDGF-A and TGF-β expression in vascular smooth muscle, but a role in megakaryocyte gene expression has not been previously suggested. To better understand the regulation of Egr-1 in erythromegakaryocytic differentiation, Nab2, a natural co-repressor of Egr-1 was also evaluated. Control K562 cells strongly expressed c-myc and Nab2. ChIP studies show that c-myc binds to the Nab2 promoter in control K562 cells and both c-myc and Nab2 expression strongly declined during the first 24 hours after PKC stimulation. Enforced expression of Nab2 not only blocked CD9 induction by IDB, but also induced marked hemoglobinization of K562 cells. Taken together, these findings suggest a novel and critical role for the balance in Egr-1/Nab2 activity as a regulatory mechanism in erythroid versus megakaryocytic differentiation pathways.

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.

Down-Regulation of Human NDR Gene in Megakaryocytic Differentiation of Erythroleukemia K562 Cells

To study the control of hematopoietic cell differentiation, a human negative differentiation regulator (NDR) gene was identified by the comparative analysis of differentially expressed genes in hemato-lymphoid tissues. NDR is expressed preferentially in the adult bone marrow, fetal liver and testis. Immunocytochemistry with anti-NDR antiserum showed the presence of NDR in human erythroleukemia K562 cell line and CD34⁺ cells sorted from the umbilical cord blood. When fused to the green fluorescent protein (GFP), NDR was directed to the nucleus of mouse 3T3 and K562 cells. Fusion protein with a deletion from residues 7 to 87 was detected in the cytoplasm. NDR appeared not to affect the proliferation of K562 cells when overly expressed. However, its expression was down-regulated during megakaryocytic differentiation of K562 cells induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Down-regulation of NDR correlated well with up-regulation of megakaryocytic markers, CD41 and CD61. Overexpression of the nuclear NDR-GFP in K562 cells inhibited the expression of CD41 and CD61 in megakaryocytic differentiation. Treatment of K562 cells with GF-109203X (GFX), an antagonist of the protein kinase C (PKC), blocked NDR down-regulation, up-regulated expression of CD41/CD61 and TPA-induced megakaryocytic differentiation. These results suggest a novel function of nuclear NDR protein in regulating hematopoietic cell development.

Transient posttranslational up-regulation of telomerase activity during megakaryocytic differentiation of K562 cells

Telomerase is active in immature somatic cells, but not in differentiated cells. However, the mechanism by which telomerase is regulated in relation to cell differentiation is not well understood. In this study, the human erythroid leukemia cell line K562 was induced to differentiate into megakaryocytes by TPA and into erythroid by STI571. The human acute myeloblastic leukemia cell line HL60 was also induced to differentiate into monocytes by TPA. Telomerase activity, the expression of human telomerase reverse transcriptase, hTERT, and the cell cycle were examined. TPA induced a transient increase in telomerase activity during the megakaryocytic differentiation while the message of hTERT decreased gradually throughout the same period. This suggests the existence of a regulatory mechanism other than transcription of hTERT. Cell cycle analysis revealed that cells in G 2/M phase increased in number in accordance with the changes in telomerase activity. Pretreatment with PKC inhibitors inhibited the megakaryocytic differentiation, transient increase in telomerase activity, and G 2/M arrest. These results suggest that PKC acts as a transient post-translational activator of telomerase during megakaryocytic differentiation.

Down-regulation of human NDR gene in megakaryocytic differentiation of erythroleukemia K562 cells.

To study the control of hematopoietic cell differentiation, a human negative differentiation regulator (NDR) gene was identified by the comparative analysis of differentially expressed genes in hemato-lymphoid tissues. NDR is expressed preferentially in the adult bone marrow, fetal liver and testis. Immunocytochemistry with anti-NDR antiserum showed the presence of NDR in human erythroleukemia K562 cell line and CD34+ cells sorted from the umbilical cord blood. When fused to the green fluorescent protein (GFP), NDR was directed to the nucleus of mouse 3T3 and K562 cells. Fusion protein with a deletion from residues 7 to 87 was detected in the cytoplasm. NDR appeared not to affect the proliferation of K562 cells when overly expressed. However, its expression was down-regulated during megakaryocytic differentiation of K562 cells induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Down-regulation of NDR correlated well with up-regulation of megakaryocytic markers, CD41 and CD61. Overexpression of the nuclear NDR-GFP in K562 cells inhibited the expression of CD41 and CD61 in megakaryocytic differentiation. Treatment of K562 cells with GF-109203X (GFX), an antagonist of the protein kinase C (PKC), blocked NDR down-regulation, up-regulated expression of CD41/CD61 and TPA-induced megakaryocytic differentiation. These results suggest a novel function of nuclear NDR protein in regulating hematopoietic cell development.

Involvement of CREB in the transcriptional regulation of the human GM3 synthase (hST3Gal V) gene during megakaryocytoid differentiation of human leukemia K562 cells

We studied the transcriptional regulation of human GM3 synthase (hST3Gal V) during megakaryocytic differentiation of K562 cells induced by PMA. Northern blot and reverse transcription polymerase chain reaction (RT-PCR) indicated that the induction of hST3Gal V by phorbol 12-myristate 13-acetate (PMA) is regulated at transcriptional level. To elucidate the mechanism underlying the regulation of the hST3Gal V gene expression during the differentiation of K562 cells induced by PMA, we characterized the promoter region of the hST3Gal V gene. Functional analysis of the 5 ′-flanking region of the hST3Gal V gene by transient expression method showed that the −177 to −83 region, which contains a CREB binding site at −143, functions as the PMA-inducible promoter in K562 cells. In addition, gel shift assay and site-directed mutagenesis indicated that the CREB binding site at −143 is crucial for the PMA-induced expression of the hST3Gal V in K562 cells.

Involvement of nucleophosmin/B23 in TPA-induced megakaryocytic differentiation of K562 cells

Human myelogenous leukaemia K562 cells were induced to undergo megakaryocytic differentiation by treatment with phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (20 nM, 24–72 h). The steady-state level of nucleophosmin/B23 mRNA decreased during the TPA-induced differentiation. There was also decrease in the level of cellular nucleophosmin/B23 protein and appearance of its degraded product (25 kDa) during the TPA-induced differentiation. Furthermore, K562/B23 (wild type), K562/D1 (Δ280–294) and K562/D2 (Δ263–294) cells were less, while K562/D3 (Δ244–294) cells were more responsive to TPA-induced differentiation as compared to K562/vector or parental K562 cells. Activation of the ERK/MAPK was observed in parental K562 cells upon TPA treatment (5 nM, 5–30 min). As compared to K562/vector cells, less activation of ERK/MAPK was observed in K562/D2 cells, while ERK/MAPK was highly activated in K562/D3 cells upon TPA treatment. Our results indicate that nucleophosmin/B23 plays an important role in TPA-induced differentiation of K562 cells and the amino acids 244–294 at C-terminal of nucleophosmin/B23 could be an important site for regulation of cellular response to differentiation.

Disabled-2 small interfering RNA modulates cellular adhesive function and MAPK activity during megakaryocytic differentiation of K562 cells

Previous studies have shown that Disabled-2 (DAB2) is up-regulated during megakaryocytic differentiation of human K562 cells. To delineate the consequences of DAB2 induction, a DNA vector-based small interfering RNA (siRNA) was designed to intervene in DAB2 expression. We found that DAB2 siRNA specifically inhibited DAB2 induction, resulting in the modulation of cell–cell adhesion and mitogen-activated protein kinase (MAPK) phosphorylation. The morphological changes and β3 integrin expression associated with megakaryocytic differentiation were not affected. Since the MAPK pathway has been shown to involve DAB2 induction [Tseng et al., Biochem. Biophys. Res. Commun. 285 (2001) 129–135], our results suggest a reciprocal regulation between DAB2 and MAPK in the differentiation of K562 cells. In addition, we have demonstrated for the first time that DAB2 siRNA is a valuable tool for unveiling the biological consequences of DAB2 expression.

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.

Expression of insulin-like growth factors IGF-I and IGF-II, and their receptors during the growth and megakaryocytic differentiation of K562 cells

Insulin-like growth factors (IGFs) I and II are critical regulators of cell proliferation and differentiation and most of the growth promoting properties of both ligands are mediated by IGF-I receptor (IGF-IR). In the present study we have investigated the role of IGFs in K562 cell line during normal growth and 12- O-tetradecanoyl-phorbol-13-acetate (TPA)-induced megakaryocytic differentiation. Abundant expression of IGF-I, IGF-II and IGF-IR was demonstrated in resting cells and exogenous IGF-I and IGF-II increased 3 H -thymidine incorporation in a dose dependent manner. In contrast, we found that basal growth of the cells was inhibited by using anti-IGF-IR mAb. Furthermore, also IGF-I and IGF-II induced DNA synthesis was significantly suppressed by anti-IGF-IR mAb. During megakaryocytic differentiation, expression of IGF-IR increased during first 12 h, but after that the expression started to decrease together with IGF-I. Taken together, our data suggest that autocrine production of IGF-I and IGF-II may via IGF-IR play a significant role in the growth and megakaryocytic differentiation of K562 cells.

Induction of Disabled-2 Gene during Megakaryocyte Differentiation of K562 Cells

Megakaryocyte differentiation is often accompanied by the changes of gene expression pattern. Here we reported that the expression of DAB2, a putative adaptor protein in cell signaling, was induced at the protein and mRNA levels upon 12-O-tetradecanoylphorbol-13-acetate-mediated megakaryocyte differentiation of human chronic myeloid leukemic K562 cells. On the other hand, the differentiation agents DMSO and retinoic acid had no effect on DAB2 expression. Analysis of promoter activity with the human DAB2 luciferase reporter constructs suggested that the regulation is partially at the transcriptional level. The responsive sequences located within an 80-bp DAB2 promoter region. To determine the involvement of MEK1-p42/p44 MAPK pathway in mediating DAB2 gene expression, we have performed the following experiments and found that (i) there was sustained activation of p42/p44 MAPK, but not p38 MAPK, upon K562 cells differentiation; (ii) application of MEK1 inhibitor U0126 reduced the expression of DAB2 protein, mRNA and promoter activity, as well as cell differentiation; (iii) constitutively active MEK1 increased DAB2 promoter activity; and (iv) dominant negative ERK2 abolished constitutively active MEK1-induced DAB2 promoter activity. Taken together, our results indicate that DAB2 gene is induced upon megakaryocyte differentiation by the MEK1-p42/p44 MAPK pathway and may define a new role of DAB2 in hematopoietic cell differentiation.

Extracellular Signal-regulated Kinase/90-kDa Ribosomal S6 Kinase/Nuclear Factor-κB Pathway Mediates Phorbol 12-Myristate 13-Acetate-induced Megakaryocytic Differentiation of K562 Cells

Two signaling pathways, the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK)-dependent pathway and the nuclear factor-κB (NF-κB)-dependent pathway, have been known to mediate megakaryocytic differentiation of K562 cells induced by phorbol 12-myristate 13-acetate (PMA). In this study, we examined whether 90-kDa ribosomal S6 kinase (RSK), known as a substrate of ERK/MAPK and a signal-inducible IκBα kinase, would link two pathways during the differentiation. RSK1 was activated in a time- and dose-dependent manner during the PMA-induced differentiation. Overexpression of wild-type or dominant inhibitory mutant (D205N) of RSK1 enhanced or suppressed PMA-stimulated NF-κB activation and megakaryocytic differentiation as shown by morphology, nonspecific esterase activity, and expression of the CD41 megakaryocytic marker, respectively. In addition, overexpression of the dominant inhibitory mutant (S32A/S36A) of IκBα inhibited PMA-stimulated and RSK1-enhanced megakaryocytic differentiation, indicating that NF-κB mediates a signal for megakaryocytic differentiation downstream of RSK1. PMA-stimulated activation of ERK/MAPK, RSK1, and NF-κB and the PMA-induced megakaryocytic differentiation were prevented by pretreatment with PD98059, a specific inhibitor of the mitogen-activated ERK kinase (MEK). Therefore, these results demonstrate that the sequential ERK/RSK1/NF-κB pathway mediates PMA-stimulated megakaryocytic differentiation of K562 cells.

Nerve growth factor potentiated the sodium butyrate- and PMA-induced megakaryocytic differentiation of K562 leukemia cells

We have recently reported that retinoic acid (RA) induced the expression of trkA, the high affinity receptor for nerve growth factor (NGF), in human chronic myelogenous leukemia K562 cells. In this paper, we examined the ability of several other differentiation inducers to regulate the expression of trkA and NGF in K562 cells. We found that the expression of trkA was dramatically induced by the two megakaryocyte lineage inducers sodium butyrate (NaBut) and phorbol 12-myristate 13-acetate (PMA), but not by the two erythrocyte lineage inducers hemin or 1-β- d-arabinofuranosyl cytosine (Ara-C). Furthermore, activation of the up-regulated trkA receptor by exogenous NGF potentiated the megakaryocytic differentiation of K562 cells induced by NaBut and PMA. Our results demonstrated that trkA is one of the essential genes that are up-regulated and involved in the megakaryocytic differentiation of K562 leukemia cells triggered by these differentiation inducers. Our findings suggest that NGF, in addition to its pivotal roles in the nervous system, may also play important roles in hematopoietic differentiation.