Search

Myelodysplastic syndrome (MDS) is characterised by ineffective erythropoiesis and poor progenitor response to erythropoietin (Epo). The aim of this study was to determine the role of the Epo-R mediated signalling in the riseof MDS and whether alteration of signalling pathways contribute to the leukeamogenesis from MDS to acute leukaemia. We analysed Epo and GM-CSF induced ERK1/2 activation, c-Fos expression, STAT-5 and AP-1 DNA binding activities in mononuclear cells of umbilical cord blood (UCBMNC), normal marrow (NBMMNC) or marrow with MDS, AML with prior MDS and de novo AML. In UCBMNC and NBMMNC, Epo and GM-CSF induced the activation of STAT-5 DNA binding and ERK1/2 activation (n = 6). In contrast, in MDS RA, both signalling pathways were activated only by GM-CSF but not by Epo (n = 7). In acute leukaemia, elevated basal activity of STAT-5 DNA binding appeared in 8/8 cases, which was independent of Epo or GM-CSF treatment. In normal and MDS samples, c-Fos and Egr-1 proteins were not detectable and the expression levels were not increased by Epo or GM-CSF treatment. In contrast, we found an elevated level of c-Fos expression in 5/8 acute leukemia cases, which was not further increased in the presence of Epo or GM-CSF. The elevated c-Fos expression was accompanied by an extremely high blast number in 5/5 cases. These results suggest that impaired ERK/MAPK activation, similarlytoimpaired STAT-5activation in Epo-R signalling, may be responsible for the apoptotic process and the block of maturation in MDS RA. The results also suggest that the appearance of the constitutively activated STAT-5 DNA binding and c-Fos expression may be used as a predictor of the blastic transformation. [ABSTRACT FROM AUTHOR]

Calpain has long been implicated in the regulation of cell cycle, mostly based on studies with inhibitors that lack strict specificity toward the enzyme. Further, previous work has primarily focused on one particular point, the G(1) checkpoint, and made no attempt at dissecting the full cycle in terms of calpain action. To extend and complement these findings, we tested the effect of a specific inhibitor, PD 150606, on granulocyte-macrophage-colony stimulating factor (GM-CSF)-stimulated human TF-1 cells by flow cytometry following single- and double labelling by propidium iodide and bromodeoxyuridine. Using a new algorithm of analysis, we determined the time-dependence of the absolute number of cells leaving G(1), S and G(2)M phases following the application of the inhibitor. Our results point to the simultaneous involvement of calpain activity in promoting the cycle at the G(1) checkpoint and somewhere in the G(2)M compartment. Furthermore, the inhibitor significantly impedes the progress of cells through the S phase, indicating calpain activity in S phase checkpoint signalling. Overall, our analysis suggests that calpain regulates the cell cycle at more points than previously thought.

The effect of erythropoietin (Epo) on the expression of mitogen-activated protein kinase (MAPK) target genes egr-1 and c-fos was investigated in Epo-responsive murine erythroblastic cell line ELM-I-1. Epo induced a transient rise in egr-1 mRNA without a similar effect on c-fos expression. The induction of egr-1 correlated with a rapid ERK1/2 phosphorylation and was prevented with MEK1/2 inhibitors PD 98059 and UO126. The p38 inhibitor SB 203580 enhanced ERK1/2 phosphorylation and egr-1 mRNA levels. Longer incubations of ELM-I-1 cells with Epo revealed a second later phase of increase in egr-1 expression which was also prevented by MEK1/2 inhibitors, whereas SB 203580 had a stimulatory effect. In contrast, the beta-globin mRNA production was enhanced in the presence of PD 98059 and UO126 and reduced by SB 203580. The results suggest a regulatory role of egr-1 expression in Epo signal transduction and provide pharmacological evidence for the negative modulation of differentiation-specific gene expression by the ERK1/2 pathway in murine erythroleukemia cells.

Changes in the cytoplasmic calcium concentration ([Ca(2+)](i)) regulate a wide variety of cellular processes. Here we demonstrate that increased [Ca(2+)](i) was able to induce hormone-independent survival and proliferation, as well as to evoke apoptosis in human myelo-erythroid GM-CSF/IL-3 dependent leukemia cells (TF-1). Cellular responses induced by elevated [Ca(2+)](i) depended on the duration and amplitude of the calcium-signal. Moderate or high, but transient, elevation of [Ca(2+)](i) caused a transient, biphasic activation of ERK1/2 and protected cells from hormone withdrawal-induced apoptosis.(1) In contrast, high and long-lasting elevation of [Ca(2+)](i) led to sustained activation of the ERK1/2 kinases and apoptosis of TF-1 cells. Our data suggest that a time-dependent action of the MAPK pathway works as a decision-point between cell proliferation and apoptosis.

Keratinocytes have the ability to kill pathogenic fungi and bacteria by producing antimicrobial substances. Recent studies suggest that microbial components use signaling molecules of the human Toll-like receptor (TLR) family to transduce signals in various cells. Here we provide evidence that keratinocytes express both TLR2 and TLR4 at the mRNA and protein levels, and show that TLR2 and TLR4 are present in the normal human epidermis in vivo and that their expression is regulated by microbial components. The expression of myeloid differentiation protein gene (MyD88), which is involved in the signaling pathway of many TLR, was also demonstrated in keratinocytes. LPS + IFN-gamma increased the expression of TLR2 and TLR4 50- and 5-fold respectively. Treatment of keratinocytes with Candida albicans, mannan, Mycobacterium tuberculosis or LPS with IFN-gamma resulted in the activation and nuclear translocation of NF-kappaB. Inhibition of NF-kappaB blocked the Candida-killing activity of keratinocytes, suggesting that the antimicrobial effect of keratinocytes requires NF-kappaB activation. LPS + IFN-gamma, C. albicans (4 Candida/KC), peptidoglycan (1 micro g/ml) or M. tuberculosis extract significantly increased IL-8 gene expression after 3 h of treatment (P < 0.05). The increases over the 0-h level were 15-, 8-, 10.8- and 7-fold, respectively. The microbial compound-induced increase in IL-8 gene expression could be inhibited by anti-TLR2 and anti-TLR4 neutralizing antibodies, suggesting that TLRs are involved in the pathogen-induced expression of this pro-inflammatory cytokine. Our findings stress the importance of the role of keratinocytes as a component of innate immunity.

Survival and proliferation of cells of a human myelo-erythroid CD34+ leukemia cell line (TF-1) depend on the presence of granulocyte-macrophage colony-stimulating factor or interleukin-3. Upon hormone withdrawal these cells stop proliferating and undergo apoptotic process. In this report we demonstrate that a controlled increase in [Ca2+]i induces hormone-independent survival and proliferation of TF-1 cells. We found that moderate elevation of [Ca2+]i by the addition of cyclopiasonic-acid protected TF1 cells from apoptosis. Furthermore, a higher, but transient elevation of [Ca2+]i by ionomycin treatment induced cell proliferation. In both cases caspase-3 activity was reduced, and Bcl-2 was up-regulated. Higher elevation of [Ca2+]i by ionomycin induced MEK-dependent biphasic ERK1/2 activation, sufficient to move the cells from G0/G1 to S/M phases. Meanwhile, activation of ERK1/2, phosphorylation of the Elk-1 transcription factor, and, consequently, a substantial elevation of Egr-1 and c-Fos levels and AP-1 DNA binding were observed. Moderate elevation of [Ca2+]i, on the other hand, caused a delayed monophasic activation of ERK1/2 and Elk-1 that was accompanied with only a small increase of Egr-1 and c-Fos levels and AP-1 DNA binding. The specific MEK-1 kinase inhibitor, PD98059, inhibited all the effects of increasing [Ca2+]i, indicating that the MAPK/ERK pathway activation is essential for TF-1 cell survival and proliferation. Based on these results we suggest that the elevation of the [Ca2+]i may influence the cytokine dependence of hemopoietic progenitors and may contribute to pathological hematopoiesis.

Background: In this study, the effect of antigen-presenting cells (APC), peptide concentration, and CD28 costimulation on calcium signaling, induced by antigen-specific T-cell activation, was studied by flow cytometry., Methods: We used two experimental approaches, which differed in their time scale and in the duration of the T cell-APC interaction, to measure the increase of intracellular free calcium levels ([Ca(2+)](i)) in activated T cells: (1) Fluo-3-loaded T cells were activated by cocentrifugation with peptide-loaded APC and the kinetics of fluorescence intensity changes was monitored continuously and (2) peptide-loaded APC and T cells were mixed, cocultured, and the fluorescence intensity was measured at various time intervals., Results: The calcium signal of T cells was dependent on the APC as demonstrated by the ratio of cells exhibiting high versus low fluorescence intensity and by the magnitude of the calcium signal in the activated population. Short-term interaction of T cells with less potent APC or with efficient APC in the presence of low antigen concentration resulted in decreased calcium signaling. CD28-mediated costimulation enhanced the magnitude and sustained the increase of intracellular calcium levels. In line with the strong and sustained calcium signals, the activation of the calcium-dependent transcription factors NF-AT, AP-1, and NF-kappaB was induced., Conclusions: Flow cytometric methods, feasible for the rapid and flexible analysis of calcium signaling upon antigen-specific T-cell activation, were established. Kinetics of the increase of mean fluorescence intensity reflected the calcium response of the total cell population whereas statistical analysis of fluorescence intensity at selected time points provided information on the activation state of single cells., (Copyright 2002 Wiley-Liss, Inc.)

The involvement of MAPK pathways in differentiation, proliferation and survival was investigated by comparing Epo and GM-CSF signalling in human factor-dependent myeloerythroid TF-1 cells with abnormal Epo-R. GM-CSF withdrawal induced cell-cycle arrest and apoptosis accompanied by increased caspase-3 activity, DNA degradation and reduced expression of the antiapoptotic Bcl-2 and Bcl-xl proteins. Readministration of GM-CSF but not Epo reversed these processes and induced proliferation. The GM-CSF promoted cell survival and proliferation correlated with MEK-1 dependent ERK1/2, Elk-1 and CREB phosphorylation and Egr-1, c-Fos expression as well as with increased STAT-5, AP-1, c-Myb and NF-kappaB DNA-binding. In contrast, Epo failed to activate the Raf-1/ERK1/2 MAPK pathway or to induce Egr-1 and/or c-Fos expression, while it induced erythroid differentiation in GM-CSF-deprived cells. In addition, the Epo-induced haemoglobin production was inhibited in the presence of GM-CSF. These results demonstrate that the activation of MAPK cascade is not necessary for Epo-induced haemoglobin production in TF-1 cells and suggest a negative cross-talk between the signalling of GM-CSF-stimulated cell proliferation and Epo-induced erythroid differentiation.

Erythropoietin (Epo) is the principal regulator of the production of circulating erythrocytes by controlling the proliferation, the differentiation and the survival of the erythroid progenitor cells. Early down-regulation of c-myb expression in erythroleukemia cells is a common feature of the action of Epo and chemical inducers of differentiation such as DMSO. Previously we have shown that in our Epo-responsive murine erythroleukemia cell line ELM-I-1, [Ca2+]i increasing agents can mimic the effect of Epo on c-myb expression and activate nuclear signal transduction processes involved in the induction of hemoglobin synthesis. These results also indicated that the Ca2+-induced down-regulation of c-myb expression and hemoglobin synthesis are mediated by the Ca2+/calmodulin dependent serine/threonine-specific protein phosphatase PP2B, calcineurin, but the Epo induced processes are not mediated by PP2B. In spite of this, we demonstrated in this paper that in ELM-I-1 cells the Epo-induced down-regulation of c-myb expression and hemoglobin production can be effectively enhanced by the simultaneously added [Ca2+]i-increasing agent, cyclopiazonic acid (CPA). This observation further supports the existence of at least two independent signalling pathways in the mechanism of Epo and [Ca2+]i increasing agents and the strong correlation between c-myb expression and hemoglobin production in differentiating cells. Although the c-AMP-response element binding protein (CREB) could be the common target of both calcium-dependent and -independent dephosphorylation, our results do not support the involvement of CREB in the regulation of c-myb gene expression. In addition to the calcineurin mediated down-regulation of c-myb expression, we have found a negative regulatory effect in the Ca2+-mediated transcriptional activation of certain genes. In response to [Ca2+]i-increasing agents in ELM-I-1 cells, both, egr-1 and c-fos mRNA expression increased significantly after the inhibition of calcineurin by cyclosporine A. Cyclosporin A exerted stimulatory effects on the egr-1 and c-fos expression also at lower (150-400 nM) intracellular Ca2+ levels. This potential co-regulation of c-myb, egr-1 and c-fos expression by calcineurin suggests that the negative modulation of egr-1 and c-fos expression may also be important for the induction of erythroid differentiation by [Ca2+]i-increasing agents. This negative modulation may also contribute to the Epo-induced differentiation in the case of a moderate increase of [Ca2+]i.

The Ca2+-induced expression of the primary response genes egr-1 and c-fos was investigated in the murine erythroleukaemia cell line ELM-I-1. Exposure of the cells to the Ca2+-ionophore A23187 led to a rapid transient rise in egr-1 and c-fos mRNA production followed by an increase in Egr-1 and c-Fos protein levels as well as an increase in Egr-1 and activator protein 1 (AP-1) DNA-binding activity. Preincubation of the cells with KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinases, strongly decreased the Ca2+-mediated expression of egr-1 and c-fos. In contrast, treatment with cyclosporin A, which inhibits the Ca2+/calmodulin-dependent protein phosphatase 2B or calcineurin, increased both egr-1 and c-fos mRNA production and the DNA-binding activity of the Egr-1 and AP-1 transcription factors in response to the intracellular Ca+ concentration ([Ca2+]i)-increasing agents A23187 or cyclopiazonic acid. Enhancement of the Ca2+-induced c-fos and egr-1 expression by cyclosporin A was correlated with the capability of this agent to inhibit calcineurin phosphatase activity in ELM-I-1 cells. Studies on the phosphorylation state and DNA-binding activity of the cAMP response element-binding protein (CREB) did not demonstrate an early Ca2+-dependent activation of this transcription factor, suggesting that the regulation of c-fos and egr-1 expression by Ca2+ is not linked to CREB in the haematopoietic ELM-I-1 cells. The results indicate that calcineurin exerts negative regulatory effects on both egr-1 and c-fos expression in murine erythroleukaemia cells, in addition to the calcineurin-mediated down-regulation of c-myb expression observed previously in this cell system. This study therefore emphasizes the important role of calcineurin as a negative modulator of gene expression in certain cell types.

The glycoprotein hormone, erythropoietin is the principal regulator of the production of circulating erythrocytes by controlling proliferation, differentiation and survival of its target erythroid progenitor cells. The receptor for erythropoietin is a type I cytokine receptor lacking intrinsic tyrosine kinase activity. It mediates tyrosine phosphorylation through its association with nonreceptor tyrosine kinases such as JAK2 and initiates a cascade of signalling events in response to erythropoietin. Significant progress has been made in identifying signalling pathways triggered by erythropoietin. However, the exact signalling mechanisms mediating the known physiological effects of erythropoietin in erythroid progenitor cells are poorly understood. There are many open questions including the role of Ca2+ in erythropoietin induced signal transduction. Although the results concerning the effect of erythropoietin on [Ca2+]i in various erythroid cells are conflicting, [Ca2+]i-increasing agents mimic the effect of erythropoietin on c-myb expression and activate the program of haemoglobin synthesis in murine erythroleukemia cells. An attempt is made in this review to survey recent data on the erythropoietin-induced signal transduction with respect to the different physiological effects of this hormone.

In a Hungarian family with triosephosphate isomerase (TPI) deficiency, two compound heterozygote brothers were found with the same severe decrease in TPI activity, but only one of them had the classical symptoms. In search for the pathogenesis of the differing phenotype of the same genotypic TPI deficiency, an increase in red cell membrane fluidity was found. There were roughly 100% and 30% more 16:0/20:4 and 18:0/20:4 diacyl-phosphatidylcholine species in erythrocytes from the two TPI-deficient brothers than in the probes from healthy controls. The activities of acethylcholinesterase and calmodulin induced Ca2+ ATPase were significantly enhanced in erythrocytes from the propositus as compared with those of the neurologically symptom-free brother and other members of the TPI-deficient family as well as to those from healthy controls. Both enzymes are crucially involved in the function of nerve cells. The observed differences in membrane fluidity and enzyme activities between the erythrocytes from the phenotypically differing TPI-deficient brothers underline the importance of investigations into the effect of biophysical changes in the lipid environment of the membrane proteins on the development of disseminated focal neurological disorders of unknown pathogenic origin.

Rabbits immunized with dinitrophenylated calmodulin produced monospecific antibody against CaM. Using the purified antibody, enzyme-linked immunosorbent assays (ELISAs) were carried out for calmodulin (CaM). The immunological heterogeneity of human and bovine CaMs from erythrocytes was investigated by means of indirect and inhibition ELISAs. The cross-reactivity between the two CaMs was found to be 50% in the indirect ELISA. An eight times higher concentration of human CaM was necessary to produce 30% inhibition in an inhibition assay. The effect of anti-bovine CaM on the stimulation of the red cell membrane calcium pump by human and bovine CaM has also been studied. We have found that (a) the human and bovine CaMs showed indistinguishable activator activities; (b) the antibody partially inhibited the stimulating effects of CaMs; (c) the inhibition was much less effective in the case of stimulation by human CaM. These results suggest that there is a difference in the reactivity of the anti-bovine CaM antibody with bovine and human CaMs. This difference can be attributed to a slight deviation in the antibody binding structure of the two mammalian CaMs in or near the antigenic site of the CaMs.

In a mixed endoplasmic and surface-type membrane vesicle preparation from human platelets the polyphosphoinositides PIP and PIP2, similarly to IP3, were found to induce a rapid calcium release reaction. At physiological (resting) cytoplasmic calcium concentrations (0.1-0.3 microM) the PIP2 and IP3 concentrations producing half-maximum calcium release were similar (0.7 microM) and both agents could mobilize about 30-40% of the intravesicular calcium. However, the phosphodiesteric degradation of PIP2 in the membrane vesicles was found to be negligible and the ion- and drug-sensitivities of the calcium release reactions were different. The IP3-induced calcium release was selectively inhibited by micromolar calcium concentrations and by cinnarizine, while the PIP2-induced release was blocked by magnesium ions and neomycin. The calcium release evoked by either agent was inhibited by low concentrations of lanthanum but, in contrast to the ATP-dependent calcium pump, it was insensitive to vanadate, quercetin and to the lowering of the incubation temperature. When added simultaneously or in a rapid succession, maximum effective IP3 and PIP2 concentrations produced an additive calcium release reaction. Based on these data we suggest that IP3 and PIP2, respectively, induce rapid transmembrane calcium movements involving different transport pathways and/or membrane calcium pools, which are not related to the active calcium transport systems.

In human platelets thrombin-induced calcium release from intracellular stores, the consequent influx of extracellular calcium, as well as their role in the aggregation and ATP-secretion reactions were examined. In indo-1-loaded platelets intracellular calcium release was studied in the presence of excess EGTA in the incubation medium, while calcium influx was followed after a rapid repletion of external calcium. After thrombin-stimulation both calcium release and calcium influx produced about the same peak levels of cytoplasmic free calcium but in the first case it was only a transient response, while in the latter one a sustained calcium signal was observed. Increased calcium influx could be evoked for several minutes after the addition of thrombin, it was selectively inhibited by Mg2+ (20 mM) and Ni2+ (1 mM) ions, by neomycin and by PCMB, a non-penetrating SH-group reagent. This calcium influx was practically insensitive to organic calcium channel blockers. Thrombin-induced platelet aggregation was only partial in the absence of external calcium, even if excess magnesium was present in the media, while the aggregation response became complete if external calcium was repleted. A significantly reduced aggregation could be seen in calcium-containing media if calcium influx was selectively inhibited. Platelet ATP-secretion under the same conditions did not depend on external calcium or on calcium influx. These data indicate that in thrombin-stimulated platelets the opening of specific plasma membrane calcium channels can be selectively modulated and these channels play a major role in the development of a full-scale aggregation.