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.     

Decrease in expression of alpha 5 beta 1 integrin during neuronal differentiation of cortical progenitor cells

Neuronal differentiation of embryonic neural progenitor cells is regulated by both intrinsic and extrinsic signals. Since dynamic changes in cell shape typify neuronal differentiation, cell adhesion molecules could be relevant to this process. Although it has been reported that fibronectin-integrin interactions are important for the proliferation of neural progenitor cells, little is known about the contribution of integrins to neuronal differentiation. In order to address this shortfall, we examined integrin expression on cortical progenitor cells by using immunohistochemistry and FACS analysis of cells in which GFP expression was driven by regulatory (promoter) regions of the nestin gene (nestin-GFP(+)). We here report that high levels of nestin promoter activity correlated with high expression levels of alpha(5)beta(1) integrin (alpha(5)beta(1)(high) cells). FACS analysis of nestin-GFP(+) cortical cells revealed an additional subpopulation with reduced expression of alpha(5)beta(1) integrin (alpha(5)beta(1)(low) cells). The size of the alpha(5)beta(1)(low) subpopulation increased during cortical development. To investigate the correlation between integrin and neuronal differentiation, nestin-GFP(+) cortical progenitor cells were sorted into alpha(5)beta(1)(high) or alpha(5)beta(1)(low) populations, and each potential to differentiate was analyzed. We show that the nestin-GFP(+) alpha(5)beta(1)(high) population corresponded to broadly multipotential neural progenitor cells, whereas nestin-GFP(+) alpha(5)beta(1)(low) cells appeared to be committed to a neuronal fate. These findings suggest that alpha(5)beta(1) expression on cortical progenitor cells is developmentally regulated and its downregulation is involved in the process of neuronal differentiation.     

Substantial changes of cellular iron homeostasis during megakaryocytic differentiation of K562 cells

We investigated the remodeling of iron metabolism during megakaryocytic development of K562 cells. Differentiation was successfully verified by increase of the megakaryocytic marker CD61 and concomitant decrease of the erythroid marker γ-globin. The reduction of erythroid properties was accompanied by changes in the cellular iron content and in the expression of proteins regulating cellular iron homeostasis. Independent of available inorganic or transferrin-bound extracellular iron, total intracellular iron increases while the iron-to-protein ratio decreases. The iron exporter ferroportin is downregulated within 1-6 h, followed by downregulation of transferrin receptor-1 (TfR1) and ferritin heavy chain (H-ferritin) mainly after 24-48 h. The hemochromatosis protein-1, a ligand of TfR1, peaked after 24 h. All effects were independent of iron supply with the exception of H-ferritin, which was restored by excess iron. While alterations of CD61, TfR1 and ferritin expression were revoked by a protein kinase C inhibitor, downregulation of ferroportin remained unaffected.     

The effect of differentiation inducers on the integrin expression of K562 erythroleukemia cells.

We studied the effects of differentiation-inducers on the integrin profile and adhesive properties of K562 leukemia cells. The fibronectin (Fn) receptor integrin, α₅β₁, was the only integrin expressed in suspension cultured K562 cells. When the cells were exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA) immunoreactivity for the β₁ integrin subunit was slightly enhanced. TPA exposure also induced the appearance of the α₂, α₃, α_v and β₃ integrin subunits, but the platelet integrin subunit α_IIb was not detected. On the other hand, hemin chloride-induced erythroid differentiation of K562 cells diminished the expression of the α₅β₁ integrin on the surface of the cells. Adhesion experiments with TPA-exposed K562 cells indicated that although the adherence to the extracellular matrix (ECM) proteins as a rule was low a few cells spread on these proteins. The present results specify the effects of differentiation inducers on the integrin profile of K562 cells and excludes the comprehension that TPA would induce expression of the platelet integrin α_IIb on their surface. Our results also show, that an increased expression of a certain integrin does not necessarily lead to a comparable adhesion ability on its ligand in vitro.     

Erythroid and megakaryocytic differentiation of K562 erythroleukemic cells by monochloramine

Abstract

The induction of leukemic cell differentiation is a hopeful therapeutic modality. We studied the effects of monochloramine (NH₂Cl) on erythroleukemic K562 cell differentiation, and compared the effects observed with those of U0126 and staurosporine, which are known inducers of erythroid and megakaryocytic differentiation, respectively. CD235 (glycophorin) expression, a marker of erythroid differentiation, was significantly increased by NH₂Cl and U0126, along with an increase in cd235 mRNA levels. Other erythroid markers such as γ-globin and CD71 (transferrin receptor) were also increased by NH₂Cl and U0126. In contrast, CD61 (integrin β3) and CD42b (GP1bα) expression, markers of megakaryocytic differentiation, was increased by staurosporine, but did not change significantly by NH₂Cl and U0126. NH₂Cl retarded cell proliferation without a marked loss of viability. When ERK phosphorylation (T202/Y204) and CD235 expression were compared using various chemicals, a strong negative correlation was observed (r = ?0.76). Paradoxically, NH₂Cl and staurosporine, but not U0126, induced large cells with multiple or lobulated nuclei, which was characteristic to megakaryocytes. NH₂Cl increased the mRNA levels of gata1 and scl, decreased that of gata2, and did not change those of pu.1 and klf1. The changes observed in mRNA expression were different from those of U0126 or staurosporine. These results suggest that NH₂Cl induces the bidirectional differentiation of K562. Oxidative stress may be effective in inducing leukemic cell differentiation.     

Constitutive c-myb expression in K562 cells inhibits induced erythroid differentiation but not tetradecanoyl phorbol acetate-induced megakaryocytic differentiation.

K562 cells were stably transfected with a plasmid vector constitutively expressing a full-length human c-myb gene. Parental cells possess the dual potential of inducibility of cellular differentiation along two lineages, i.e., erythroid and megakaryocytic. The resulting lineage is dependent on the inducing agent, with a number of compounds being competent to various degrees for inducing erythroid differentiation, while the tumor promoter tetradecanoyl phorbol acetate (TPA) induces a macrophage-like morphology with enhanced expression of proteins associated with megakaryocytes. Exogeneous expression of c-myb in transfected cell lines abrogated erythroid differentiation induced by cadaverine or cytosine arabinoside as assessed by hemoglobin production. However, TPA-induced megakaryocytic differentiation was left intact, as assessed by cell morphology, cytochemical staining, and the expression of the megakaryocytic antigens. These results indicate that c-Myb and protein kinase C play important roles in cellular differentiation of K562 cells and suggest that agents which directly modulate protein kinase C can induce differentiation in spite of constitutively high levels of c-Myb.     

Induction of expression of the alpha v beta 1 and alpha v beta 3 integrin heterodimers during retinoic acid-induced neuronal differentiation of murine embryonal carcinoma cells

All-trans-retinoic acid, an endogenous morphogen, induced neuronal differentiation of P19 murine embryonal carcinoma cells. Peak differentiation, as judged by the elaboration of neuronal processes, occurred 8 days after exposure of the cells to 0.5 mM retinoic acid, a concentration known to induce neuronal differentiation. An examination of the expression of the extracellular matrix receptors, integrins, during this retinoic acid-induced differentiation period, demonstrated a specific and strong induction of expression of two polypeptides (130 and 115 kDa) immunoprecipitated with an anti-human vitronectin receptor antiserum. The expression of a 90-kDa polypeptide, also immunoprecipitating with this antiserum was induced as well, but to a much smaller extent. The expression of a 96-kDa polypeptide immunoprecipitated by this antiserum and present in the untreated cells was not induced by retinoic acid. The increase in the expression of these polypeptides paralleled the neuronal differentiation of the P19 embryonal carcinoma cells. The expression of these integrins was not induced in a variant of the P19 cells, P19RAC65, which are resistant to differentiation induction by retinoic acid. Utilizing integrin subunit-specific anti-cytoplasmic peptide antibodies together with immunoprecipitation and Western blot analysis, the 130- and 115-kDa polypeptides were identified as the integrin alpha v and beta 1 subunits, respectively. The 90-kDa polypeptide, also induced by retinoic acid, was identified as beta 3, whereas the identity of the uninduced 96-kDa polypeptide remains unclear as yet. Peptide map analysis of deglycosylated polypeptides demonstrated that the 90- and 96-kDa polypeptides are distinct proteins and that the 115-kDa polypeptides immunoprecipitated with either anti-alpha v or anti-beta 1 antibodies are identical, further establishing that the 115-kDa polypeptide associating with alpha v is beta 1. The retinoic acid-induced expression of beta 1 occurred at the level of mRNA expression which also paralleled neuronal differentiation, but peaked slightly ahead of the cell surface expression of beta 1. The expression of other beta 1-associated alpha subunits was not induced by retinoic acid in these cells. These data demonstrate that retinoic acid strongly induces the expression of the integrin heterodimer alpha v beta 1 and also, to a smaller extent, the expression of alpha v beta 3. The retinoic acid-induced, high level surface expression of the alpha v beta 1 heterodimer is tightly correlated with the induction of neuronal differentiation by retinoic acid. This finding suggests an important role for the alpha v beta 1 heterodimer in the neuronal differentiation process.     

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 beta3 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.     

Essential role for ALCAM gene silencing in megakaryocytic differentiation of K562 cells

Background  

Activated leukocyte cell adhesion molecule (ALCAM/CD166) is expressed by hematopoietic stem cells. However, its role in hematopoietic differentiation has not previously been defined.     

ADAM15 suppresses cell motility by driving integrin alpha5beta1 cell surface expression via Erk inactivation

Human ADAM15 is unique among the A disintegrin and metalloprotease domain (ADAM) family because of the integrin binding motif Arg-Gly-Asp (RGD) within its disintegrin domain. Integrin alpha5beta1 has been reported to bind to ADAM15 in an RGD-dependent manner, but the biological significance of the interaction between ADAM15 and alpha5beta1 is unknown. To characterize the effects of ADAM15 on alpha5beta1-mediated cell adhesion and migration and elucidate the potential mechanism, CHO cells which express endogenous integrin alpha5beta1 were transfected with human ADAM15 cDNA. ADAM15 overexpression led to enhanced cell adhesion and decreased migration on fibronectin, which were suppressed by down-regulation of integrin alpha5. Overexpression of ADAM15 not only increased the cell surface expression of integrin alpha5 but also resulted in a more clustered staining of alpha5 on cell surface, while the beta1 subunit remained unchanged. Unexpectedly, results from immunoprecipitation and immunofluorescence indicated that ADAM15 and alpha5beta1 integrin did not interact directly in CHO cells. We found that ADAM15 expression decreased the phosphorylation of Erk1/2. Consistently, down-regulation of Erk1/2 phosphorylation by MEK inhibitor PD98059 or siRNA against Erk1/2 enhanced the expression of alpha5 on cell surface. By using a B16F10 pulmonary metastasis model, we revealed that overexpression of ADAM15 significantly reduced the number of metastatic nodules on the lung. Taken together, this study reveals for the first time that ADAM15 could drive alpha5 integrin expression on cell surface via down-regulation of phosphorylated Erk1/2. This presents a novel mechanism by which ADAM15 regulates cell-matrix adhesion and migration.