Mária Magócsi

Activation of Raf/ERK1/2 MAP kinase pathway is involved in GM-CSF-induced proliferation and survival but not in erythropoietin-induced differentiation of TF-1 cells.

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.

Dual β-adrenergic modulation in the immune system : Stimulus-dependent effect of isoproterenol on MAPK activation and inflammatory mediator production in macrophages

This is the first study to demonstrate that the interaction between beta-adrenoceptor activation, and the production of inflammatory mediators can be modulated in opposite ways by two inflammatory stimuli, namely, protein kinase C (PKC)-activating phorbol myristyl acetate (PMA) and lipopolysaccharide (LPS). We provided evidence that isoproterenol treatment, when combined with phorbol ester increased the production of tumor necrosis factor-alpha, interleukin-12, and nitric oxide in murine macrophages, as well as in human monocytes and differentiated PLB-985 cells, while in agreement with earlier findings, it decreased inflammatory mediator production in combination with LPS stimulation. The contrasting effect on inflammatory mediator production, shown for the PMA and LPS activated cells was accompanied by parallel changes in activation of ERK1/2 and p38 MAPKs. Thus, isoproterenol significantly increased MAPK activation (phosphorylation) in PMA-treated cells and, conversely, it decreased the activation of extracellular signal regulated kinase 1/2 (ERK1/2) and p38 in LPS-stimulated cells. The opposing effects of isoproterenol on LPS-induced versus PMA-induced mediator production and the concurrent changes in MAPK activation highlight the role of this kinase pathway in macrophage activation and provide new insights regarding the flexible ways through which beta-adrenoceptor stimulation can modulate the inflammatory response in macrophages. Our results challenge the dogma that beta-adrenoceptor signaling is only immunosuppressive, and offer potential opportunities for new therapeutic approaches in the treatment of inflammatory and autoimmune diseases.

Signalling Mechanisms in Erythropoiesis: The Enigmatic Role of Calcium

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.

Multiple G‐protein‐coupling specificity of β‐adrenoceptor in macrophages

Adrenergic signalling of the immune system is one of the important modulator pathways of the inflammatory immune response realized via G protein‐mediated pathways. The resulted signal depends on the type of the receptor‐coupled G‐protein (GPCR) that, according to the classical paradigm in the case of β‐adrenergic receptor (β‐AR), is Gs‐type. Recently, alternate and/or multiple G protein coupling specificity of GPCRs have been demonstrated including a switch from Gs to Gi binding. The possibility of a Gs/Gi switch and its role in the immune response of macrophages has not been investigated yet. In this study, we demonstrate that β‐adrenergic stimulation itself is able to induce a transient mitogen‐activated protein kinase phosphorylation in murine peritoneal macrophages in a pertussis toxin‐sensitive manner, suggesting that the Gs/Gi switch also occurs in the immune system. Although this process is very rapid, it can influence different signalling pathways and can reprogramme effector functions suggesting that sympathetic modulation of the defence mechanism of the innate immune system has an additional, Gs/Gi switch‐dependent component.

Ca2+/Calmodulin-dependent and -independent Down-regulation of c-myb mRNA Levels in Erythropoietin-responsive Murine Erythroleukemia Cells THE ROLE OF CALCINEURIN

Down-regulation of c-myb mRNA levels by [Ca2+]i-increasing agents (A23187, thapsigargin, cyclopiazonic acid) and erythropoietin was comparatively studied in the erythropoietin-responsive murine erythroleukemia cell line, ELM-I-1. The Ca2+-induced suppression of c-myb mRNA could be inhibited by the calmodulin antagonists trifluoperazine and calmidazolium, as well as by cyclosporin A, an inhibitor of the Ca2+/calmodulin-dependent protein phosphatase 2B (calcineurin). KN-62, an inhibitor of Ca2+/calmodulin-dependent protein kinases, did not antagonize the Ca2+-mediated decrease in c-myb mRNA. In cyclosporin A-treated ELM-I-1 cells, a close correlation could be demonstrated between the antagonization of the Ca2+ effect on c-myb mRNA levels and inhibition of the calcineurin phophatase activity. On the other hand, FK506, which did not inhibit calcineurin activity in ELM-I-1 cells, failed to prevent the Ca2+-mediated decrease in c-myb mRNA. The erythropoietin-induced down-regulation of c-myb mRNA levels could be demonstrated also in the presence of EGTA and was resistant to calmodulin antagonists and cyclosporin A. In addition, no increase in [Ca2+]i was observed in ELM-I-1 cells in response to erythropoietin. Cyclosporin A inhibited the Ca2+-induced hemoglobin production, while the erythropoietin-mediated increase in hemoglobin synthesis was not affected. The results indicate that the Ca2+-induced decrease in c-myb mRNA and increase in hemoglobin synthesis is mediated by calcineurin, while these effects of erythropoietin occur independently of Ca2+ in ELM-I-1 cells. Calcineurin may be involved in the regulation of c-myb expression in erythroid precursor cells and Ca2+ signals via calcineurin may positively modulate the differentiation inducing action of erythropoietin.

IL-7 withdrawal induces a stress pathway activating p38 and Jun N-terminal kinases

IL-7 delivers survival signals to cells at an early stage in lymphoid development. In the absence of IL-7, pro-T cells undergo programmed cell death, which has previously been associated with a decline in Bcl-2 and translocation of Bax from cytosol to mitochondria. A new, earlier feature of IL-7 withdrawal was identified using an IL-7-dependent thymocyte line. We observed that withdrawal of IL-7 induced increased expression of jun and fos family member genes including c-jun, junB, junD, c-fos and fra2. This transient response peaked 3-4 h after IL-7 was withdrawn and resulted in increased DNA-binding activity of AP-1 and in a change in the composition of the Jun/Fos family dimers shown by electrophoretic mobility shift and supershift assays. Induction of jun and fos genes and the increased DNA-binding activity of AP-1 were attributable to the phosphorylation-induced activation of the stress kinases p38 and JNK and were blocked by the chemical kinase inhibitors SB203580 and SB202190. The stress response contributed to cell death following IL-7 withdrawal as shown by blocking the activity of the stress (MAP) kinases or by blocking the production of c-Jun and c-Fos using antisense oligonucleotides.

Effects of intracellular calcium on cell survival and the MAPK pathway in a human hormone-dependent leukemia cell line (TF-1).

Abstract: Changes in the cytoplasmic calcium concentration ([Ca2+]i) regulate a wide variety of cellular processes. Here we demonstrate that increased [Ca2+]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 [Ca2+]i depended on the duration and amplitude of the calcium‐signal. Moderate or high, but transient, elevation of [Ca2+]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 [Ca2+]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.

Unregulated activation of STAT-5, ERK1/2 and c-Fos may contribute to the phenotypic transformation from myelodysplastic syndrome to acute leukaemia.

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 rise of 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 ERK 1/2 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, similarly to impaired STAT-5 activation 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.