Versatility in signalling: multiple responses to EGF receptor activation during Drosophila oogenesis

The Drosophila epidermal growth factor receptor (EGFR) is active in different tissues and is involved in diverse processes such as patterning of the embryonic ectoderm, growth and differentiation of imaginal discs and cell survival. During oogenesis, the EGFR is expressed in the somatic follicle cells that surround individual oocyte-nurse cell complexes. In response to germline signals, the follicle cells differentiate in a complex pattern, which in turn leads to the establishment of the egg axes. Two recent reports have shown that the strategies used to pattern posterior follicle cells are different from those used to pattern dorsal follicle cells. In posterior follicle cells, EGFR activity is translated into an on-off response, whereas, in dorsal follicle cells, patterning mechanisms are initiated and refined by feedback that modulates receptor activity over time.     

HGF/MET signalling protects Plasmodium-infected host cells from apoptosis

Plasmodium, the causative agent of malaria, migrates through several hepatocytes before initiating a malaria infection. We have previously shown that this process induces the secretion of hepatocyte growth factor (HGF) by traversed cells, which renders neighbour hepatocytes susceptible to infection. The signalling initiated by HGF through its receptor MET has multifunctional effects on various cell types. Our results reveal a major role for apoptosis protection of host cells by HGF/MET signalling on the host susceptibility to infection. Inhibition of HGF/MET signalling induces a specific increase in apoptosis of infected cells leading to a great reduction on infection. Since HGF/MET signalling is capable of protecting cells from apoptosis by using both PI3-kinase/Akt and, to a lesser extent, MAPK pathways, we determined the impact of these pathways on Plasmodium sporozoite infection. Although inhibition of either of these pathways leads to a reduction in infection, inhibition of PI3-kinase/Akt pathway caused a stronger effect, which correlated with a higher level of apoptosis in infected host cells. Altogether, the results show that the HGF/MET signalling requirement for infection is mediated by its anti-apoptotic signal effects. These results demonstrate for the first time that active inhibition of apoptosis in host cell during infection by Plasmodium is required for a successful infection.     

Hedgehog activates the EGF receptor pathway during Drosophila head development.

The Hedgehog (Hh) and Epidermal growth factor receptor (EGFR) signaling pathways play critical roles in pattern formation and cell proliferation in invertebrates and vertebrates. In this study, we demonstrate a direct link between these two pathways in Drosophila melanogaster. Hh and EGFR signaling are each required for the formation of a specific region of the head of the adult fruitfly. We show that hh and vein (vn), which encodes a ligand of the Drosophila EGFR (Schnepp, B., Grumbling, G., Donaldson, T. and Simcox, A. (1996) Genes Dev. 10, 2302-13), are expressed in adjacent domains within the imaginal primordium of this region. Using loss- and gain-of-function approaches, we demonstrate that Hh activates vn expression. We also show that Hh activation of vn is mediated through the gene cubitus interruptus (ci) and that this activation requires the C-terminal region of the Ci protein. Finally, we demonstrate that wingless (wg) represses vn expression, thereby limiting the domain of EGFR signaling.     

The EGF receptor and N signalling pathways act antagonistically in Drosophila mesothorax bristle patterning

An early step in the development of the large mesothoracic bristles (macrochaetae) of Drosophila is the expression of the proneural genes of the achaete-scute complex (AS-C) in small groups of cells (proneural clusters) of the wing imaginal disc. This is followed by a much increased accumulation of AS-C proneural proteins in the cell that will give rise to the sensory organ, the SMC (sensory organ mother cell). This accumulation is driven by cis-regulatory sequences, SMC-specific enhancers, that permit self-stimulation of the achaete, scute and asense proneural genes. Negative interactions among the cells of the cluster, triggered by the proneural proteins and mediated by the Notch receptor (lateral inhibition), block this accumulation in most cluster cells, thereby limiting the number of SMCs. Here we show that the proneural proteins trigger, in addition, positive interactions among cells of the cluster that are mediated by the Epidermal growth factor receptor (EGFR) and the Ras/Raf pathway. These interactions, which we denominate 'lateral co-operation', are essential for macrochaetae SMC emergence. Activation of the EGFR/Ras pathway appears to promote proneural gene self-stimulation mediated by the SMC-specific enhancers. Excess EGFR signalling can overrule lateral inhibition and allow adjacent cells to become SMCs and sensory organs. Thus, the EGFR and Notch pathways act antagonistically in notum macrochaetae determination.     

Phospholipid membrane composition affects EGF receptor and Notch signaling through effects on endocytosis during Drosophila development

The role of phospholipids in the regulation of membrane trafficking and signaling is largely unknown. Phosphatidylcholine (PC) is a main component of the plasma membrane. Mutants in the Drosophila phosphocholine cytidylyltransferase 1 (CCT1), the rate-limiting enzyme in PC biosynthesis, show an altered phospholipid composition with reduced PC and increased phosphatidylinositol (PI) levels. Phenotypic features of dCCT1 indicate that the enzyme is not required for cell survival, but serves a role in endocytic regulation. CCT1- cells show an increase in endocytosis and enlarged endosomal compartments, whereas lysosomal delivery is unchanged. As a consequence, an increase in endocytic localization of EGF receptor (Egfr) and Notch is observed, and this correlates with a reduction in signaling strength and leads to patterning defects. A further link between PC/PI content, endocytosis, and signaling is supported by genetic interactions of dCCT1 with Egfr, Notch, and genes affecting endosomal traffic.     

Autophagy protects breast cancer cells from epirubicin-induced apoptosis and facilitates epirubicin-resistance development

Epirubicin (EPI) is one of the most effective drugs against cancer. But the acquired resistance of cancer cells to EPI is becoming a major obstacle for successful cancer therapy. Recently, some studies have revealed that macroautophagy (here referred to as autophagy) may protect the cancer cell from anticancer drug-induced death, so autophagy might be related to the development of drug resistance to these reagents. However, the relationship between autophagy and drug resistance has yet to be defined. Our study showed that EPI induced autophagy in human breast cancer MCF-7 cells. And the EPI-induced autophagy protected MCF-7 cells from EPI-induced apoptosis. Furthermore, autophagy was elevated in EPI-resistant MCF-7 cells (MCF-7er cells), and inhibition of autophagy restored the sensitivity of MCF-7er cells to EPI. Therefore, autophagy is a prosurvival factor and has a role in the development of EPI-acquired resistance in EPI-treated MCF-7 cells. Also, this finding indicates that the use of clinically applicable autophagy inhibitors might be one of the important strategies for breast cancer therapy.     

Autophagy protects breast cancer cells from epirubicin-induced apoptosis and facilitates epirubicin-resistance development

Epirubicin (EPI) is one of the most effective drugs against cancer. But the acquired resistance of cancer cells to EPI is becoming a major obstacle for successful cancer therapy. Recently, some studies have revealed that macroautophagy (here referred to as autophagy) may protect the cancer cell from anticancer drug-induced death, so autophagy might be related to the development of drug resistance to these reagents. However, the relationship between autophagy and drug resistance has yet to be defined. Our study showed that EPI induced autophagy in human breast cancer MCF-7 cells. And the EPI-induced autophagy protected MCF-7 cells from EPI-induced apoptosis. Furthermore, autophagy was elevated in EPI-resistant MCF-7 cells (MCF-7er cells), and inhibition of autophagy restored the sensitivity of MCF-7er cells to EPI. Therefore, autophagy is a prosurvival factor and has a role in the development of EPI-acquired resistance in EPI-treated MCF-7 cells. Also, this finding indicates that the use of clinically applicable autophagy inhibitors might be one of the important strategies for breast cancer therapy.     

ARAP1 regulates EGF receptor trafficking and signalling

The activation state of the EGF receptor (EGF-R) is tightly controlled in the cell so as to prevent excessive signalling, with the dangerous consequences that this would have on cell growth and proliferation. This control occurs at different levels, with a key level being the trafficking and degradation of the EGF-R itself. Multiple guanosine triphosphatases belonging to the Arf, Rab and Rho families and their accessory proteins have key roles in these processes. In this study, we have identified ARAP1, a multidomain protein with both Arf GTPase-activating protein (GAP) and Rho GAP activities, as a novel component of the machinery that controls the trafficking and signalling of the EGF-R. We show that ARAP1 localizes to multiple cell compartments, including the Golgi complex, as previously reported, and endosomal compartments, where it is enriched in the internal membranes of multivesicular bodies. ARAP1 distribution is controlled by its phosphorylation and by its interactions with the 3- and 4-phosphorylated phosphoinositides through its five PH domains. We provide evidence that ARAP1 controls the late steps of the endocytic trafficking of the EGF-R, with ARAP1 knockdown leading to EGF-R accumulation in a sorting/late endosomal compartment and to the inhibition of EGF-R degradation that is accompanied by prolonged signalling.     

EGF receptor signalling: the importance of presentation

Activation of the Drosophila EGF receptor requires the transmembrane TGF-alpha-like ligand Spitz. Recent studies have shed new light on the role of two transmembrane proteins, Star and Rhomboid, in the presentation and subsequent proteolytic processing of Spitz.     

Neurotensin protects pancreatic beta cells from apoptosis

The survival of pancreatic beta cells depends on the balance between external cytotoxic and protective molecular systems. The neuropeptide neurotensin (NT) has been shown to regulate certain functions of the endocrine pancreas including insulin and glucagon release. However, the mechanism of action of NT as well as the identification of receptors involved in the pancreatic functions of the peptide remained to be studied. We demonstrate here that NT is an efficient protective agent of pancreatic beta cells against cytotoxic agents. Both beta-TC3 and INS-1E cell lines and the mouse pancreatic islet cells express the three known NT receptors. The incubation of beta cells with NT protects cells from apoptosis induced either by staurosporine or by IL-1beta. In beta-TC3 cells, NT activates both MAP and PI-3 kinases pathways and strongly reduces the staurosporine or the Il-1beta-induced caspase-3 activity by a mechanism involving Akt activation. The NTSR2 agonist levocabastine displays the same protective effect than NT whereas the NTSR1 antagonist is unable to block the effect of NT suggesting the predominant involvement of the NTSR2 in the action of NT on beta cells. These results clearly indicate for the first time that NT is able to protect endocrine beta cells from external cytotoxic agents, a role well correlated with its release in the circulation after a meal.     

Caspase inhibition during apoptosis causes abnormal signalling and developmental aberrations in Drosophila

Programmed cell death or apoptosis plays an important role in the development of multicellular organisms and can also be induced by various stress events. In the Drosophila wing imaginal disc there is little apoptosis in normal development but X-rays can induce high apoptotic levels, which eliminate a large fraction of the disc cells. Nevertheless, irradiated discs form adult patterns of normal size, indicating the existence of compensatory mechanisms. We have characterised the apoptotic response of the wing disc to X-rays and heat shock and also the developmental consequences of compromising apoptosis. We have used the caspase inhibitor P35 to prevent the death of apoptotic cells and found that it causes increased non-autonomous cell proliferation, invasion of compartments and persistent misexpression of the wingless (wg) and decapentaplegic (dpp) signalling genes. We propose that a feature of cells undergoing apoptosis is to activate wg and dpp, probably as part of the mechanism to compensate for cell loss. If apoptotic cells are not eliminated, they continuously emit Wg and Dpp signals, which results in developmental aberrations. We suggest that a similar process of uncoupling apoptosis initiation and cell death may occur during tumour formation in mammalian cells.     

The EGF receptor and notch signaling pathways control the initiation of the morphogenetic furrow during Drosophila eye development

The onset of pattern formation in the developing Drosophila retina begins with the initiation of the morphogenetic furrow, the leading edge of a wave of retinal development that transforms a uniform epithelium, the eye imaginal disc into a near crystalline array of ommatidial elements. The initiation of this wave of morphogenesis is under the control of the secreted morphogens Hedgehog (Hh), Decapentaplegic (Dpp) and Wingless (Wg). We show that the Epidermal Growth Factor Receptor and Notch signaling cascades are crucial components that are also required to initiate retinal development. We also show that the initiation of the morphogenetic furrow is the sum of two genetically separable processes: (1) the 'birth' of pattern formation at the posterior margin of the eye imaginal disc; and (2) the subsequent 'reincarnation' of retinal development across the epithelium.     

Activated EGF receptor may balance ERK-inhibitory network signalling pathways

In the COS-7 cell signalling network high levels of cAMP produced, for example, by co-stimulation of beta2-adrenergic receptor (beta2-AR) and bradykinin B2 receptor (BKR) may affect epidermal growth factor receptor (EGFR)-mediated activation of extracellular signal-stimulated kinase (ERK). In contrast, co-stimulation of either beta2-AR or B2R with EGFR leads to synergistic activation of ERK. Due to triple stimulation of these receptors the synergistic effects on ERK activation as well as cAMP accumulation are diminished. Here we demonstrate that EGF is capable of inducing Src-mediated phosphorylation of the tyrosine residues 177 and 347 of BKR. Their replacement by phenylalanine led to BKR mutants which are unable to activate the cAMP pathway. Using these mutants we can show that EGF attenuates but does not completely inhibit the BKR/cAMP pathway which is counteracting the EGFR signalling to ERK. Our findings suggest that the EGFR may control the cellular network rather by balancing mechanisms then by switch on/off reactions.     

MK2-dependent p38b signalling protects Drosophila hindgut enterocytes against JNK-induced apoptosis under chronic stress

The integrity of the intestinal epithelium is crucial for the barrier function of the gut. Replenishment of the gut epithelium by intestinal stem cells contributes to gut homeostasis, but how the differentiated enterocytes are protected against stressors is less well understood. Here we use the Drosophila larval hindgut as a model system in which damaged enterocytes are not replaced by stem cell descendants. By performing a thorough genetic analysis, we demonstrate that a signalling complex consisting of p38b and MK2 forms a branch of SAPK signalling that is required in the larval hindgut to prevent stress-dependent damage to the enterocytes. Impaired p38b/MK2 signalling leads to apoptosis of the enterocytes and a subsequent loss of hindgut epithelial integrity, as manifested by the deterioration of the overlaying muscle layer. Damaged hindguts show increased JNK activity, and removing upstream activators of JNK suppresses the loss of hindgut homeostasis. Thus, the p38/MK2 complex ensures homeostasis of the hindgut epithelium by counteracting JNK-mediated apoptosis of the enterocytes upon chronic stress.     

The transduction signalling protein Go during embryonic development of Drosophila melanogaster

G proteins are heterotrimeric proteins that play a key role in signalling transduction conveying signals from cell surface receptors to intracellular effector proteins. In particulate preparations from Drosophila melanogaster embryos, only one substrate of 39,000-40,000 molecular weight could be ADP-ribosylated with pertussis toxin. This substrate reacted in immunoblotting and immunoprecipitation experiments with a polyclonal antibody directed against the carboxy-terminal sequence of the alpha subunit of the mammalian Go protein. The Drosophila Go alpha protein was present at all stages of embryonic development; however, its expression markedly increased after 10 h embryogenesis, a period of time during which there is an active development of axonal tracts. Immunolocalization on whole mount embryos has indicated that this protein is principally localized in the CNS and is mainly restricted to the neuropil without any labelling of the cell bodies. In contrast, all the axon tracts of the CNS appeared to be highly labelled. The distribution of the Go alpha protein was also examined in several neurogenic mutants. The Go alpha protein expression was not altered in any of them but the pattern of labelling was disorganized as was the neuronal network. These results suggest a possible role for the Go protein during axonogenesis.