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A plethora of extracellular stimuli regulate growth, survival, and differentiation responses through activation of the MEK-ERK MAPK signaling module. Using CD34+ hematopoietic progenitor cells, we describe a novel role for the MEK-ERK signaling module in the regulation of proliferation, survival, and cytokine production during neutrophil differentiation. Addition of the specific MEK1/2 inhibitor U0126 resulted in decreased proliferation of neutrophil progenitors. Conversely, transient activation of a conditionally active MEK1 mutant resulted in the expansion of progenitor cells, which thereafter differentiated normally into mature neutrophils. In contrast, chronic MEK1 activation was found to induce cell death of CD34+ neutrophil progenitors. Microarray analysis of CD34+ progenitor cells showed that activation of MEK1 resulted in changes in expression of a variety of cell-cycle modulating genes. Furthermore, conditional activation of MEK1 resulted in a dramatic increase in the expression of mRNA transcripts encoding a large number of hematopoietic cytokines, chemokines, and growth factors. These findings identify a novel role for MEK-ERK signaling in regulating the balance between proliferation and apoptosis during neutrophil differentiation, and they suggest the need for tight control of MEK-ERK activation to prevent the development of bone marrow failure.

Survival signals elicited by cytokines include the activation of phosphatidylinositol 3-kinase (PI3K), which in turn promotes the activation of protein kinase B (PKB). Recently, PKB has been demonstrated to phosphorylate and inactivate forkhead transcription factor FKHR-L1, a potent inducer of apoptosis. To explore the mechanisms underlying the induction of apoptosis after cytokine withdrawal or FKHR-L1 activation, we used a cell line in which FKHR-L1 activity could be specifically induced. Both cytokine withdrawal and FKHR-L1 activation induced apoptosis, which was preceded by an upregulation in p27KIP1 and a concomitant decrease in cells entering the cell cycle. Induction of apoptosis by both cytokine withdrawal and activation of FKHR-L1 correlated with the disruption of mitochondrial membrane integrity and cytochrome c release. This was preceded by upregulation of the pro-apoptotic Bcl-2 family member Bim. Ectopic expression of an inhibitory mutant of FKHR-L1 substantially reduced the levels of apoptosis observed after cytokine withdrawal. Activation of PKB alone was sufficient to promote cell survival, as measured by maintenance of mitochondrial integrity and the resultant inhibition of effector caspases. Furthermore, hematopoietic stem cells isolated from Bim-/- mice exhibited reduced levels of apoptosis upon inhibition of PI3K/PKB signaling. These data demonstrate that activation of FKHR-L1 alone can recapitulate all known elements of the apoptotic program normally induced by cytokine withdrawal. Thus PI3K/PKB--mediated inhibition of this transcription factor likely provides an important mechanism by which survival factors act to prevent programmed cell death.

Background: Cardiopulmonary bypass (CPB) surgery initiates a controlled systemic inflammatory response characterized by a cytokine storm, monocytosis and transient monocyte activation. However, the responsiveness of monocytes to Toll-like receptor (TLR)-mediated activation decreases throughout the postoperative course. The purpose of this study was to identify the major signaling pathway involved in plasma-mediated inhibition of LPS-induced tumor necrosis factor (TNF)-α production by monocytes. Methodology/Principal Findings: Pediatric patients that underwent CPB-assisted surgical correction of simple congenital heart defects were enrolled (n = 38). Peripheral blood mononuclear cells (PBMC) and plasma samples were isolated at consecutive time points. Patient plasma samples were added back to monocytes obtained pre-operatively for ex vivo LPS stimulations and TNF-α and IL-6 production was measured by flow cytometry. LPS-induced p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB activation by patient plasma was assessed by Western blotting. A cell-permeable peptide inhibitor was used to block STAT3 signaling. We found that plasma samples obtained 4 h after surgery, regardless of pre-operative dexamethasone treatment, potently inhibited LPS-induced TNF-α but not IL-6 synthesis by monocytes. This was not associated with attenuation of p38 MAPK activation or IkB-α degradation. However, abrogation of the IL-10/STAT3 pathway restored LPS-induced TNF-α production in the presence of suppressive patient plasma. Conclusions/Significance: Our findings suggest that STAT3 signaling plays a crucial role in the downregulation of TNF-α synthesis by human monocytes in the course of systemic inflammation in vivo. Thus, STAT3 might be a potential molecular target for pharmacological intervention in clinical syndromes characterized by systemic inflammation. [ABSTRACT FROM AUTHOR]

Signal transducer and activator of transcription 5 (STAT5) activity is induced by a plethora of cytokines and growth factors resulting in transcriptional activation of specific target genes. STAT5 plays an important role in a variety of cellular processes, including proliferation, differentiation, and apoptosis. Aberrant regulation of STAT5 has been observed in solid tumors as well as in patients with either chronic or acute myeloid leukemia. Kinase inhibitors are currently being developed to negatively regulate STAT5 activity for clinical purposes. [Copyright &y& Elsevier]

Granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and interleukin-5 (IL-5 belong to a family of cytokines that regulate proliferation, differentiation and function of haematopoietic cells. Their receptor consists of a ligand specific α-chain and a signal transducing β-chain (βc). While, the role of phosphotyrosine residues in the βc as mediators of downstream signalling cascades has been established, little is known about non-phosphotyrosine mediated events. To identify proteins interacting with βc, we screened a yeast two-hybrid library with the intracellular domain of βc. We found that RACK1, a molecule associating with activated PKC, PLCγ and Src kinases, associated with the membrane proximal region of βc in both yeast two-hybrid, immunoprecipitation and GST-pull-down assays. The association of RACK1 was constitutive, demonstrating no alteration upon cellular stimulation. Furthermore, upon stimulation of cells with IL-5 or PMA, a complex of βc and PKCβ was found. Together, these findings suggest a novel role for RACK1 as a possible adapter molecule associating with the intracellular domain of cytokine receptors. [ABSTRACT FROM AUTHOR]

Interleukin (IL)-3, IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) regulate proliferation, differentiation and apoptosis of target cells. Receptors for these cytokines consist of a cytokine-specific α subunit and a common shared βc subunit. Tyrosine phosphorylation of the βc is thought to play a critical role in mediating signal transduction events. We have examined the effect of mutation of βc tyrosines on the activation of multiple signal transduction pathways. Activation of protein kinase B (PKB) required JAK2 and was inhibited by dominant-negative phosphatidylinositol 3-kinase (P13K). Overexpression of JAK2 was sufficient to activate both protein kinase B (PKB) and extracellular regulated kinase-1 (ERK1). Tyrosine 577 and 612 were found to be critical for the activation of PKB and ERK1, but not activation of STAT transcription factors. Activation of both PKB and ERK have been implicated in the regulation of proliferation and apoptosis. We generated GM-CSFR stable cell lines expressing receptor mutants to evaluate their effect on these processes. Activation of both PKB and ERK was perturbed, while STAT activation remained unaffected. Tyrosines 577 and 612 were necessary for optimal proliferation, however, mutation of these tyrosine residues did not affect GM-CSF mediated rescue from apoptosis. These data demonstrate that while phosphorylation of βc tyrosine residues 577 and 612 are important for optimal cell proliferation, rescue from apoptosis can be mediated by alternative signalling routes apparently independent of PKB or ERK activation. [ABSTRACT FROM AUTHOR]

During the last decade research has focused on the application of FOXP3+ regulatory T cells (Tregs) in the treatment of autoimmune disease. However, thorough functional characterization of these cells in patients with chronic autoimmune disease, especially at the site of inflammation, is still missing. Here we studied Treg function in patients with juvenile idiopathic arthritis (JIA) and observed that Tregs from the peripheral blood as well as the inflamed joints are fully functional. Nevertheless, Treg-mediated suppression of cell proliferation and cytokine production by effector cells from the site of inflammation was severely impaired, because of resistance to suppression. This resistance to suppression was not caused by a memory phenotype of effector T cells or activation status of antigen presenting cells. Instead, activation of protein kinase B (PKB)/c-akt was enhanced in inflammatory effector cells, at least partially in response to TNFα and IL-6, and inhibition of this kinase restored responsiveness to suppression. We are the first to show that PKB/c-akt hyperactivation causes resistance of effector cells to suppression in human autoimmune disease. Furthermore, these findings suggest that for a Treg enhancing strategy to be successful in the treatment of autoimmune inflammation, resistance because of PKB/c-akt hyperactivation should be targeted as well. [ABSTRACT FROM AUTHOR]

Despite the crucial function of dendritic cells (DC) in immunity, the molecular mechanisms regulating human DC development remain poorly defined. STAT5 regulates various hematopoietic lineages and is activated by GM-CSF, a critical cytokine in DC development. In this study, we investigated the role of STAT5 during differentiation of human CD34+ hematopoietic progenitors into precursor DC (pre-DC) and their subsequent differentiation toward interstitial DC and Langerhans cells. Inhibiting STAT5 activity by dominant-negative STAT5 promoted Langerhans cell commitment of hematopoietic progenitors but resulted in loss of pre-interstitial DC development, showing subset-specific regulation. Increasing the low endogenous STAT5 activity by ectopic STAT5 activation downregulated expression of the critical DC transcription factor PU.1 and abrogated commitment to either DC lineage. In contrast, high STAT5 activity was beneficial in already committed pre-DC: terminal DC differentiation was associated with increased endogenous STAT5 phosphorylation levels, JAK2-STAT5 inhibition reduced terminal DC differentiation, and conditional STAT5 activation in pre-DC improved development of BDCA-1+, DC-SIGN+, and Langerin+ DC with normal maturation and T cell stimulation. These data show that STAT5 critically regulates human DC development, with specific requirements for the level of STAT5 activation at distinct differentiation stages. By regulating STAT5 activity, cytokines present at specific locations and under different pathophysiological conditions can determine the fate of DC precursors. [ABSTRACT FROM AUTHOR]

AF18748 is a dimeric peptide of 2×19 amino acids that specifically binds to the α-chain of the human IL-5 receptor (IL-5R), preventing binding of IL-5 and acting as a receptor antagonist. However, the peptide acts by inducing α-chain dimerization, and we therefore set out to investigate whether this peptide would have any residual agonist activity. AF18748 was unable to induce activation of a number of signal transduction pathways, but was able to specifically block IL-5-dependent signal transduction. The peptide was unable to support the survival and proliferation of the cytokine dependent cell line, Ba/F3–IL-5R, and was unable to prime the respiratory burst, or induce adhesion, migration or survival of primary human eosinophils. In each case the AF18748 functioned as an antagonist. These data suggest that AF18748 may be useful to specifically modulate eosinophil function in vivo. [Copyright &y& Elsevier]

Identifies a mechanism of transcriptional activation by cytokine-specific receptor subunits. Role for the cytoplasmic domain of interleukin-5-specific alpha-subunit; Construction of a two-hybrid cDNA library from human granulocytes; Proliferation of progenitors of B1 cells.