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OBJECTIVE: The signaling pathways induced by promegapoietin (PMP), a family of chimeric growth factors that activate the human IL-3 and c-Mpl receptors, were investigated. METHODS: The biological activity of PMP was examined by receptor binding, cell proliferation, ex vivo expansion of hematopoietic progenitor cells, and in vivo production of platelets. The activation of signaling pathways was examined by Western blot and Northern blot analyses. RESULTS: Two PMP molecules, PMP-1 and PMP-1a, induced proliferation of cells expressing the IL-3 receptor, c-Mpl, or both receptors and bound to the IL-3 receptor and c-Mpl with high affinity. Ex vivo expansion assays using human bone marrow CD34(+) cells suggested that PMP-1 induced greater total cellular expansion as well as expansion of CD41(+) megakaryocytic precursor cells than IL-3 or c-Mpl ligand alone. Subcutaneous administration of 50 microg/kg of PMP-1 for 10 days to rhesus monkeys resulted in increased platelet production in vivo from a baseline of 357 +/- 45 x 10(3) cells/mL to 1376 +/- 151 x 10(3) cell/mL. PMP-1 induced phosphorylation of the beta(c) subunit of IL-3 receptor and c-Mpl, JAK2, and STAT5b, but not STAT3. PMP-1 induced greater expression of Pim-1, c-Myc, and cyclin D2 than did either an IL-3 receptor agonist or c-Mpl receptor agonist alone. The magnitude of induction of early response genes was similar for PMP and the coaddition of IL-3 receptor agonist and c-Mpl receptor agonist. CONCLUSION: PMP combines the biological activities of IL-3 and c-Mpl ligand in a single molecule that can simultaneously activate signaling pathways induced by both these ligands.
Many distinct signaling pathways allow the cell to receive, process, and respond to information. Often, components of different pathways interact, resulting in signaling networks. Biochemical signaling networks were constructed with experimentally obtained constants and analyzed by computational methods to understand their role in complex biological processes. These networks exhibit emergent properties such as integration of signals across multiple time scales, generation of distinct outputs depending on input strength and duration, and self-sustaining feedback loops. Feedback can result in bistable behavior with discrete steady-state activities, well-defined input thresholds for transition between states and prolonged signal output, and signal modulation in response to transient stimuli. These properties of signaling networks raise the possibility that information for "learned behavior" of biological systems may be stored within intracellular biochemical reactions that comprise signaling pathways.
Hypochlorous acid is an important oxidizing agent produced by neutrophils to aid in defense against pathogens. Although hypochlorous acid is known to cause tissue damage due to its cytotoxicity, the effect of this oxidizing agent on signal transduction by cells of the immune system and its effects on their responses are not well understood. In this study, hypochlorous acid was found to induce cellular tyrosine phosphorylation in both T and B lymphocytes, activate the ZAP-70 tyrosine kinase, and induce cellular calcium signaling in a tyrosine kinase-dependent manner. These signaling events also occurred in T cell lines that did not express the T-cell receptor, indicating the ability of hypochlorous acid to bypass normal receptor control. Hypochlorous acid induced tumor necrosis factor-alpha production in peripheral blood mononuclear cells in a tyrosine kinase-dependent manner. These results suggest that hypochlorous acid may contribute to inflammatory responses by activating signal pathways in cells of the immune system.
Receptors coupled to heterotrimeric G proteins are linked to activation of mitogen-activated protein kinases (MAPKs) via receptor- and cell-specific mechanisms. We have demonstrated recently that gonadotropin-releasing hormone (GnRH) receptor occupancy results in activation of extracellular signal-regulated kinase (ERK) through a mechanism requiring calcium influx through L-type calcium channels in alphaT3-1 cells and primary rat gonadotropes. Further studies were undertaken to explore the signaling mechanisms by which the GnRH receptor is coupled to activation of another member of the MAPK family, c-Jun N-terminal kinase (JNK). GnRH induces activation of the JNK cascade in a dose-, time-, and receptor-dependent manner in clonal alphaT3-1 cells and primary rat pituitary gonadotrophs. Coexpression of dominant negative Cdc42 and kinase-defective p21-activated kinase 1 and MAPK kinase 7 with JNK and ERK indicated that specific activation of JNK by GnRH appears to involve these signaling molecules. Unlike ERK activation, GnRH-stimulated JNK activity does not require activation of protein kinase C and is not blocked after chelation of extracellular calcium with EGTA. GnRH-induced JNK activity was reduced after treatment with the intracellular calcium chelator BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester), whereas activation of ERK was not affected. Chelation of intracellular calcium also reduced GnRH-induced activation of JNK in rat pituitary cells in primary culture. GnRH-induced induction and activation of the JNK target c-Jun was inhibited after chelation of intracellular calcium, whereas induction of c-Fos, a known target of ERK, was unaffected. Therefore, although activation of ERK by GnRH requires a specific influx of calcium through L-type calcium channels, JNK activation is independent of extracellular calcium but sensitive to chelation of intracellular calcium. Our results provide novel evidence that GnRH activates two MAPK superfamily members via strikingly divergent signaling pathways with differential sensitivity to activation of protein kinase C and mobilization of discrete pools of calcium.
Signal transduction in cancer cells is a sophisticated process that involves receptor tyrosine kinases (RTKs) that eventually trigger multiple cytoplasmic kinases, which are often serine/threonine kinases. A number of tumor models have identified several key cellular signaling pathways that work independently, in parallel, and/or through interconnections to promote cancer development. Three major signaling pathways that have been identified as playing important roles in cancer include the phosphatidyl inositol-3-kinase (PI3K)/AKT, protein kinase C (PKC) family, and mitogen-activated protein kinase (MAPK)/Ras signaling cascades. In clinical trials, highly selective or specific blocking of only one of the kinases involved in these signaling pathways has been associated with limited or sporadic responses. Improved understanding of the complexity of signal transduction processes and their roles in cancer has suggested that simultaneous inhibition of several key kinases at the level of receptors and/or downstream serine/threonine kinases may help to optimize the overall therapeutic benefit associated with molecularly targeted anticancer agents. Using targeted agents to inhibit multiple signaling pathways has emerged as a new paradigm for anticancer treatment based on preclinical and clinical data showing potent anti-tumor activity of single drugs inhibiting multiple molecular targets or combination therapies involving multiple drugs with selective or narrow target specificity. Preclinical and clinical studies point to molecules on vascular endothelial cells and pericytes as being important targets for anticancer therapies, as well as molecules on or within tumor cells themselves. This suggests that optimal therapeutic approaches to cancer may involve targeting multiple molecules found in both the tumor and supportive tissues. In this review, we will use the most recent preclinical and clinical data to describe this emerging paradigm for anticancer therapy involving targeting multiple signaling pathways with tyrosine or serine/threonine kinase inhibitors.
Multiple intracellular signaling pathways have been shown to regulate the hypertrophic growth of cardiomyocytes. Both necessary and sufficient roles have been described for the mitogen activated protein kinase(1) (MAPK) signaling pathway, specific protein kinase C (PKC) isoforms, and calcineurin. Here we investigate the interdependence between calcineurin, MAPK, and PKC isoforms in regulating cardiomyocyte hypertrophy using three separate approaches. Hearts from hypertrophic calcineurin transgenic mice were characterized for PKC and MAPK activation. Transgenic hearts demonstrated activation of c-Jun NH(2)-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK1/2), but not p38 MAPK factors. Calcineurin transgenic hearts demonstrated increased activation of PKCalpha, beta(1), and theta, but not of epsilon, beta(2), or lambda. In a second approach, cultured cardiomyocytes were infected with a calcineurin adenovirus to induce hypertrophy and the effects of pharmacologic inhibitors or co-infection with a dominant negative adenovirus were examined. Calcineurin-mediated hypertrophy was prevented with PKC inhibitors, Ca(2+) chelation, and attenuated with a dominant negative SEK-1 (MKK4) adenovirus, but inhibitors of ERK or p38 activation had no effect. In a third approach, we examined the activation of MAPK factors and PKC isoforms during the progression of load-induced hypertrophy in aortic banded rats with or without cyclosporine. We determined that inhibition of calcineurin activity with cyclosporine prevented PKCalpha, theta, and JNK activation, but did not affect PKCepsilon, beta, lambda, ERK1/2, or p38 activation. Collectively, these data indicate that calcineurin hypertrophic signaling is interconnected with PKCalpha, theta, and JNK in the heart, while PKCepsilon, beta, lambda, p38, and ERK1/2 are not involved in calcineurin-mediated hypertrophy.
The Notch signaling pathway regulates the commitment and early development of T lymphocytes. We studied Notch-mediated induction of the pre-T cell receptor alpha (pTa) gene, a T-cell-specific transcriptional target of Notch. The pTa enhancer was activated by Notch signaling and contained binding sites for its nuclear effector, CSL. Mutation of the CSL-binding sites abolished enhancer induction by Notch and delayed the up-regulation of pTa transgene expression during T cell lineage commitment. These results show a direct mechanism of stage- and tissue-specific gene induction by the mammalian Notch/CSL signaling pathway.
The study of signaling pathways has begun to uncover the mechanism by which cells respond and adapt to extracellular stimuli. It has become increasingly clear that the signaling pathways interact with one another to form a complex network through which regulation occurs. Here, we focus on three mechanisms by which signaling pathways interact and the physiological consequences of these interactions. Coincident signaling in long-term depression of synaptic responses in the cerebellum, protein kinase A gating of Ras to mitogen-activated protein kinase signal flow in proliferative responses, and a modified gating mechanism by phosducin resulting in feedback regulation of signal flow from rhodopsin to the cGMP phosphodiesterase in retinal light adaptation are analyzed as examples of different types of interactions between signaling pathways. These interactions allow the cell to spatially and temporally integrate complex information and respond in an appropriate and defined manner.
We examined signalling pathways which can involve NO as a second messenger in the respiratory network. In the functional slice preparation, NO donors depressed the respiratory motor output and enhanced its depression after brief episodes of hypoxia. In the inspiratory neurons, NO donors suppressed spontaneous excitatory and inhibitory synaptic currents, activated single K(ATP) channels and inhibited L-type Ca2+ channels. NO scavengers, PTIO and hemoglobin, and the blocker of NO synthase, N-monomethyl-L-arginine, induced effects opposite to those of NO donors and indicated the role of endogenously generated NO in the modulation of the respiratory activity. Using fluorescent dyes DAF-2 and DCF, we imaged NO and reactive oxygen species (ROS). Concentrations of NO and ROS increased during brief episodes of hypoxia and they both contributed to the activation of K(ATP) channels due to oxygen withdrawal. The oxidizing agent t-butyl-hydroperoxide acted similarly to NO donors but it did not interfere with the effects of NO. Increase in cGMP levels with 8-Br-cGMP reproduced the actions of NO donors and occluded the effects of their subsequent applications. We propose that in the respiratory neurons, a constitutive production of NO is responsible for a tonic activation of cGMP-coupled signalling pathways and changes in NO levels modulate the respiratory motor output by altering the activity of K(ATP) and L-type Ca2+ channels.
Acceleration of the polyol pathway and enhanced oxidative stress are implicated in the pathogenesis of diabetic complications. We and others recently reported that aldose reductase (AR), the rate-limiting enzyme in the polyol pathway, was upregulated by reactive oxygen and nitrogen species in vascular smooth muscle cells. To clarify the molecular mechanisms underlying these findings, we investigated the signal transduction pathways mediating AR expression using the rat vascular smooth muscle cell line A7r5. A selective epidermal growth factor (EGF) receptor kinase inhibitor, tyrphostin AG1478, significantly suppressed the hydrogen peroxide (H2O2)-induced increase in AR mRNA and enzyme activity. Activation of extracellular signal-regulated protein kinase (ERK) by H2O2 was blunted by AG1478. PD98059, a specific inhibitor of ERK kinase (MEK1), reduced H2O2-induced AR expression. EGF alone elicited activation of ERK and induction of AR expression. Increased level of AR transcript was demonstrated in cells treated with oxidized low-density lipoprotein, and this increase was also suppressed by AG1478. Inhibition of p38 MAP kinase by SB203580 also partially suppressed the H2O2-initiated AR induction. The presence of ponalrestat, an AR inhibitor, significantly accelerated H2O2-induced cell death. These results suggested that AR may act as a survival factor in these cells and that the EGF receptor-ERK pathway is the major signaling pathway involved in the upregulation of AR expression under oxidative stress.
Nitric oxide signaling is achieved through both cGMP-dependent and cGMP-independent mechanisms. The latter are exemplified by protein thiol modification followed by subsequent NAD(+)-dependent automodification of the glycolytic enzyme GAPDH, or by mechanisms inducing accumulation of the tumor suppressor gene p53 and causing apoptotic cell death. Both cGMP-independent actions are initiated using NO-releasing compounds and an active LPS/cytokine-inducible NO synthase. NO-synthase inhibitors block the release of NO and hinder downstream signaling mechanisms; they are therefore valuable pharmacological tools linking a defined cellular response to various NO actions. Signal transducing mechanisms elicited by NO can be studied using GAPDH as a representative example of NO-induced protein modification and are grouped as follows: --S-Nitrosylation reactions initiated by NO+ --NAD(+)-dependent, post-translational covalent automodification of GAPDH --Oxidative modification (thiol oxidation) and inhibition of GAPDH by NO-related agents, probably ONOO- GAPDH and several other protein targets may serve as molecular sensors of elevated NO concentrations and may transmit this message through posttranslational modification and oxidation-induced conformational changes as cGMP-independent NO signaling pathways. Toxicity of NO seems to be linked to both apoptosis and necrosis, depending on the chemistry of NO it undergoes in a given biological milieu. Toxicity manifests as a relative excess of NOx, metal-NO interactions, and ONOO- formation in relation to cellular defense systems. Although accumulation of the tumor-suppressor gene product p53 in response to NO opens a regulatory mechanism known to be involved in apoptotic cell death, cGMP-independent signaling pathways remain to be elucidated. As NO-dependent modification of GAPDH would imply down-regulation of glycolysis and concomitant energy production followed by cell death, our data so far do not support this assumption. In recent years, NO has proved to be a beneficial messenger with a potentially toxic activity. It will be challenging to investigate NO biochemistry in closer detail and to elucidate how NO targets biological systems, especially in relation to its pathophysiological role.
Serum and certain growth factors have the ability to inhibit programmed cell death (apoptosis) and promote survival. The mechanism by which growth factors deliver an anti-apoptotic signal and the mechanism by which this survival signal is uncoupled from mitogenesis are not clear. We studied five downstream effectors of growth factor receptors--Ras, Raf, Src, phosphoinositide 3-kinase (PI 3-kinase), and Akt (PKB)--for their abilities to block apoptosis. Activated forms of Ras, Raf, and Src, although transforming, were not sufficient to deliver a survival signal upon serum withdrawal. In contrast, inhibition of PI 3-kinase accelerated apoptosis, and an activated form of the serine/threonine kinase Akt, a downstream effector of PI 3-kinase, blocked apoptosis. The ability of Akt to promote survival was dependent on and proportional to its kinase activity. In Rat1a fibroblasts, activated Akt did not alter Bcl-2 or Bcl-X(L) expression but inhibited Ced3/ICE-like activity. Thus, the PI 3-kinase/Akt (PKB) signaling pathway transduces a survival signal that ultimately blocks Ced3/ICE-like activity. These results suggest that uncoupling of survival and mitogenesis can be explained by differing abilities of distinct mitogens to efficiently induce the PI 3-kinase/Akt signaling pathway.
The Wnt signalling pathway can activate transcription of genes such as c-myc through beta-catenin. Here, we describe the protein breakpoint cluster region, Bcr, as a negative regulator of this pathway. Bcr can form a complex with beta-catenin and negatively regulate expression of c-Myc. Knockdown of Bcr by short interfering RNA relieves the block and activates expression of c-Myc. Expression of Bcr in the human colon carcinoma cell line HCT116, which has a high level of endogenous beta-catenin, leads to reduced c-Myc expression. The negative effect is exerted by the amino terminus of Bcr, which does not harbour the kinase domain. Bcr-Abl, the oncogene protein expressed in chronic myelogenous leukaemia (CML), does not bind to beta-catenin. It phosphorylates Bcr in the first exon sequence on tyrosines, which abrogates the binding of Bcr to beta-catenin. The inhibitor of the Bcr-Abl tyrosine kinase, STI-571 or Gleevec, a drug against CML, reverses this effect. Our data contribute to the understanding of Bcr as a tumour suppressor in the Wnt signalling pathway, as well as in CML.
The Akt signaling pathway plays a crucial role in neuronal survival, leading to inhibition of apoptosis. Many stimulants including neurotrophins are reported to activate this pathway in preclinical studies; however, there are no drugs for neurodegenerative diseases adopting such a concept on the market so far. Among neurodegenerative diseases, Alzheimer's disease is the most common and characterized by senile plaques and neurofibrillary tangles, which consist of beta-amyloid and hyperphosphorylated tau, respectively. Recent studies suggest that activation of Akt inhibits toxicity of beta-amyloid and formation of neurofibrillary tangles, leading to protection of neurons against apoptosis. This review discusses the possibility of treatment of Alzheimer's disease by activating the Akt signaling pathway.
The Notch-signalling pathway is important in establishing metameric pattern during somitogenesis. In mice, the lack of either of two molecules involved in the Notch-signalling pathway, Mesp2 or presenilin-1 (Ps1), results in contrasting phenotypes: caudalized versus rostralized vertebra. Here we adopt a genetic approach to analyse the molecular mechanism underlying the establishment of rostro-caudal polarity in somites. By focusing on the fact that expression of a Notch ligand, Dll1, is important for prefiguring somite identity, we found that Mesp2 initiates establishment of rostro-caudal polarity by controlling two Notch-signalling pathways. Initially, Mesp2 activates a Ps1-independent Notch-signalling cascade to suppress Dll1 expression and specify the rostral half of the somite. Ps1-mediated Notch-signalling is required to induce Dll1 expression in the caudal half of the somite. Therefore, Mesp2- and Ps1-dependent activation of Notch-signalling pathways might differentially regulate Dll1 expression, resulting in the establishment of the rostro-caudal polarity of somites.
The signal transducing integral membrane protein, MecR1 helps initiate the expression of the antibiotic-resistant gene mecA, which encodes the penicillin-binding protein 2a. MecA participates in the beta-lactam resistance of methicillin-resistant Staphylococcus aureus (MRSA). Blocking the MecR1 regulatory pathway may be a novel strategy to combat MRSA. In this study, we introduced an antisense phosphothioate oligodeoxynucleotide (PS-ODN) targeting MecR1 mRNA into the MRSA strain WHO-2, which led to a significant reduction of both MecR1 and PBP2a mRNAs in a concentration-dependent manner. Consequently, the susceptibility of S. aureus WHO-2 to the beta-lactam antibiotic oxacillin was restored significantly. Our results indicate that blocking the mecR1-mecI-mecA signaling pathway via an antisense approach might be a viable strategy to restore the susceptibility of MRSA to the existing beta-lactam antibiotics.
Previous studies have shown that the Epstein-Barr virus-encoded latent membrane protein1 (LMP1) could activate nuclear factor kappa B, activator protein-1, and Janus kinases/signal transducer and activation of transcription factors pathways. However, many signaling molecules and downstream target proteins triggered by LMP1 have not been identified. To determine the functional components in signaling pathways triggered by LMP1, we combined the novel strategy of phosphoprotein enrichment with proteomics technology to elucidate the signaling cascade activated by LMP1. We found that LMP1 could increase the quantity of total phosphoproteins by 18.03%, and 43 proteins showed significant changes in the degree of phosphorylation when LMP1 was expressed. Twenty-five signaling molecules or downstream targets of signaling pathways triggered by LMP1 were identified, several of which had previously been implicated in LMP1 signal pathways. The other proteins, including annexin A2, heat shock protein 27, stathmin, annexin I, basic transcription factor 3, and porin, were novel signaling molecules or targets with no previously known function in LMP1 signal transduction. The method used here has proven to be suitable for the identification of molecules involved in various signaling pathways.