Balance of life and death in alveolar epithelial type II cells: proliferation, apoptosis, and the effects of cyclic stretch on wound healing

After acute lung injury, repair of the alveolar epithelium occurs on a substrate undergoing cyclic mechanical deformation. While previous studies showed that mechanical stretch increased alveolar epithelial cell necrosis and apoptosis, the impact of cell death during repair was not determined. We examined epithelial repair during cyclic stretch (CS) in a scratch-wound model of primary rat alveolar type II (ATII) cells and found that CS altered the balance between proliferation and cell death. We measured cell migration, size, and density; intercellular gap formation; cell number, proliferation, and apoptosis; cytoskeletal organization; and focal adhesions in response to scratch wounding followed by CS for up to 24 h. Under static conditions, wounds were closed by 24 h, but repair was inhibited by CS. Wounding stimulated cell motility and proliferation, actin and vinculin redistribution, and focal adhesion formation at the wound edge, while CS impeded cell spreading, initiated apoptosis, stimulated cytoskeletal reorganization, and attenuated focal adhesion formation. CS also caused significant intercellular gap formation compared with static cells. Our results suggest that CS alters several mechanisms of epithelial repair and that an imbalance occurs between cell death and proliferation that must be overcome to restore the epithelial barrier.     

Ataxia telangiectasia-mutated gene product inhibits DNA damage-induced apoptosis via ceramide synthase.

DNA double-stranded breaks (dsb) activate surveillance systems that identify DNA damage and either initiate repair or signal cell death. Failure of cells to undergo appropriate death in response to DNA damage leads to misrepair, mutations, and neoplastic transformation. Pathways linking DNA dsb to reproductive or apoptotic death are virtually unknown. Here we report that metabolic incorporation of 125I-labeled 5-iodo-2'deoxyuridine, which produces DNA dsb, signaled de novo ceramide synthesis by post-translational activation of ceramide synthase (CS) and apoptosis. CS activation was obligatory, since fumonisin B1, a fungal pathogen that acts as a specific CS inhibitor, abrogated DNA damage-induced death. X-irradiation yielded similar results. Furthermore, inhibition of apoptosis using the peptide caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone did not affect CS activation, indicating this event is not a consequence of induction of apoptosis. ATM, the gene mutated in ataxia telangiectasia, is a member of the phosphatidylinositol 3-kinase family that constitutes the DNA damage surveillance/repair system. Epstein-Barr virus-immortalized B cell lines from six ataxia telangiectasia patients with different mutations exhibited radiation-induced CS activation, ceramide generation, and apoptosis, whereas three lines from normal patients failed to manifest these responses. Stable transfection of wild type ATM cDNA reversed these events, whereas antisense inactivation of ataxia telangiectasia-mutated gene product in normal B cells conferred the ataxia telangiectasia phenotype. We propose that one of the functions of ataxia telangiectasia-mutated gene product is to constrain activation of CS, thereby regulating DNA damage-induced apoptosis.     

Effects of TGF-alpha gene knockout on epithelial cell kinetics and repair of methotrexate-induced damage in mouse small intestine

While previous studies have indicated that exogenous TGF-alpha stimulates epithelial growth, maintenance, and repair of the gut, roles of endogenous TGF-alpha are less well-defined particularly in the small bowel. The current study examined effects of TGF-alpha knockout on adult small intestinal epithelial cell proliferation, migration, apoptosis, and damage/repair response after methotrexate treatment. Compared to normal mice, TGF-alpha gene knockout did not affect crypt cell production, mitosis position, migration, and apoptosis in non-injured intestine. RT-PCR gene expression analysis revealed presence of four out of six TGF-alpha related EGF family ligands in the normal intestine, suggesting a possible functional redundancy of the EGF family in maintenance of the intestine. Although TGF-alpha gene knockout did not significantly impair the overall mucosal repair in methotrexate-induced acute damage in the small intestine, it resulted in a higher apoptotic response in the early hours following methotrexate challenge, and a delayed and reduced crypt cell proliferation during repair. Consistently, after methotrexate challenge, intestinal TGF-alpha mRNA was found to be markedly upregulated in the early hours and during repair in the wild type, and there were similar profiles in the increased expression of all other ligands (except EGF) between the wild type and knockout intestines. Therefore, despite a possible functional redundancy among the EGF family ligands in the normal small intestine, TGF-alpha may play a role in modulating the early apoptotic events and in enhancing the subsequent reparative proliferative response in the methotrexate-damaged intestine.     

The metalloproteinase ADAM‐12 regulates bronchial epithelial cell proliferation and apoptosis

Abstract. Objectives: The ADAMs (a disintegrin and metalloproteinase) enzymes compose a family of membrane‐bound proteins characterized by their multi‐domain structure and ADAM‐12 expression is elevated in human non‐small cell lung cancers. The aim of this study was to investigate the roles played by ADAM‐12 in critical steps of bronchial cell transformation during carcinogenesis. Materials and methods: To assess the role of ADAM‐12 in tumorigenicity, BEAS‐2B cells were transfected with a plasmid encoding human full‐length ADAM‐12 cDNA, and then the effects of ADAM‐12 overexpression on cell behaviour were explored. Treatment of clones with heparin‐binding epidermal growth factor (EGF)‐like growth factor (HB‐EGF) neutralizing antibodies as well as an EGFR inhibitor allowed the dissection of mechanisms regulating cell proliferation and apoptosis. Results: Overexpression of ADAM‐12 in BEAS‐2B cells promoted cell proliferation. ADAM‐12 overexpressing clones produced higher quantities of HB‐EGF in their culture medium which may rely on membrane‐bound HB‐EGF shedding by ADAM‐12. Targeting HB‐EGF activity with a neutralizing antibody abrogated enhanced cell proliferation in the ADAM‐12 overexpressing clones. In sharp contrast, targeting of amphiregulin, EGF or transforming growth factor‐α failed to influence cell proliferation; moreover, ADAM‐12 transfectants were resistant to etoposide‐induced apoptosis and the use of a neutralizing antibody against HB‐EGF activity restored rates of apoptosis to be similar to controls.Conclusions: ADAM‐12 contributes to enhancing HB‐EGF shedding from plasma membranes leading to increased cell proliferation and reduced apoptosis in this bronchial epithelial cell line.     

The protective effect of EGF-activated ROS in human corneal epithelial cells by inducing mitochondrial autophagy via activation TRPM2

Oxidative stress is a major pathogenesis of some ocular surface diseases. Our previous study demonstrated that epidermal growth factor (EGF)-activated reactive oxygen species (ROS) could protect against human corneal epithelial cell (HCE) injury. In the present study, we aimed to explore the role and mechanisms of oxidative stress and mitochondrial autophagy in HCE cells subjected to scratch injury. CCK-8 assays, EdU assays, Western blot analysis, wound-healing assays, and flow cytometry were conducted to determine cell viability, proliferation, protein expression, cell apoptosis, and intracellular ROS levels, respectively. The results showed that EGF could promote damage repair and inhibit cell apoptosis in scratch injured HCE cells by upregulating ROS (**p < .01, ***p < .001). EGF also induced mitochondrial autophagy and alleviated mitochondrial damage. Interestingly, the combination of the mitochondrial autophagy inhibitor and mitochondrial division inhibitor 1 (MDIVI-1) with EGF could reduce cell proliferation, viability, and the ROS level (*p < .05, **p < .01, ***p < .001). Treatment using the ROS inhibitor N-acetyl- l -cysteine abrogated the increase in mitochondrial membrane potential after EGF treatment. (*p < .05). Taken together, these findings indicated that EGF plays an important role in HCE damage repair and could activate ROS to protect against HCE injury by inducing mitochondrial autophagy via activation of TRPM2.     

Bcl-2 inhibits cell death of serum-free mouse embryo cells caused by epidermal growth factor deprivation

SFME cells are brain-derived neural precursor cells that are acutely dependent on epidermal growth factor (EGF) for survival, undergoing apoptosis within 24 h after EGF withdrawal. Because the expression of the protooncogene bcl-2 inhibits apoptosis induced by the withdrawal of interleukins or nerve growth factor in some growth factor-dependent haematopoietic or neuronal cell cultures, we examined the effect of Bcl-2 expression on cell death of SFME cells in the absence of EGF. SFME cells expressing human Bcl-2 showed prolonged survival when deprived of EGF compared to control cells not expressing Bcl-2. A significant fraction of Bcl-2-expressing cells remained viable for 4 days in the absence of EGF and resumed proliferation upon readdition of EGF to the cultures. These results suggest that apoptosis induced by EGF withdrawal in SFME cells may share common mechanisms with other growth factor-related apoptotic systems.     

EGF receptor phosphorylation is affected by ionizing radiation

Eukaryotic cells respond to ionizing radiation with cell cycle arrest, activation of DNA repair mechanisms, and lethality. However, little is known about the molecular mechanisms that constitute these responses. Here we report that ionizing radiation enhances epidermal growth factor (EGF) receptor tyrosine phosphorylation in intact cells as well as in isolated membranes of A431 cells. Phosphoamino acid analysis revealed that ionizing radiation preferentially enhances tyrosine phosphorylation, while EGF enhances the phosphorylation of all three phosphoamino acids (serine, threonine and tyrosine) of the EGF receptor. In addition, radiation reduces the turnover rate of the EGF receptor, while EGF increases the rate of the receptor turnover and down-regulation. Moreover, the confined radiation-induced phosphorylation of tyrosine residues is inhibited by genistein, indicating that this phosphorylation of EGF receptor is due to protein tyrosine kinase activation. These studies provide novel insights into the capacity of radiation to modulate EGF receptor phosphorylation and function. The radiation-induced elevation in the EGF receptor tyrosine phosphorylation and the receptor's slower rate of turnover are discussed in terms of their possible role in cell growth and apoptosis modulation.     

Epidermal growth factor induces cell cycle arrest and apoptosis of squamous carcinoma cells through reduction of cell adhesion

Most squamous epithelial cells are strictly anchorage-dependent cell types. We observed that epidermal growth factor (EGF) promoted the growth of A431 squamous carcinoma cells in suspension cultures but suppressed cell growth and induced apoptosis in monolayer cultures, suggesting that loss of adhesion is responsible for the effects observed in monolayer culture, before cell death. Consistent with this finding, we demonstrated that EGF reduced cell attachment, cell-cell interaction, and cell spreading. Treatment with EGF increased cell adhesion-regulated expression of p21 but suppressed expressions of cyclin A, D1, cdk2, and retinoblastoma protein (pRb), leading to cell cycle arrest and adhesion-regulated programmed cell death. To test directly whether promoting cell adhesion could reduce the effects of EGF, we grew cultures on plates coated with type II collagen. On these plates, cell adhesion was enhanced and EGF treatment had little effect on cell adhesion and apoptosis when cells were attached to the collagen. The collagen effects were dose dependent, and cell cycle and cell cycle-associated proteins were altered accordingly. Finally, when cultures were plated on bacterial Petri dishes, which completely disrupted cell attachment to substratum, the level of apoptosis was greatly higher and cell cycle was arrested as compared with monolayer cultures. Taken together, our results strongly suggest that the EGF-induced cell cycle arrest and apoptosis in monolayer cultures was the result of a decline in cell adhesion.     

EGF receptor.

The receptor for the epidermal growth factor (EGF) and related ligands (EGFR), the prototypal member of the superfamily of receptors with intrinsic tyrosine kinase activity, is widely expressed on many cell types, including epithelial and mesenchymal lineages. Upon activation by at least five genetically distinct ligands (including EGF, transforming growth factor-alpha (TGF alpha) and heparin-binding EGF (HB-EGF)), the intrinsic kinase is activated and EGFR tyrosyl-phosphorylates itself and numerous intermediary effector molecules, including closely-related c-erbB receptor family members. This initiates myriad signaling pathways, some of which attenuate receptor signaling. The integrated biological responses to EGFR signaling are pleiotropic including mitogenesis or apoptosis, enhanced cell motility, protein secretion, and differentiation or dedifferentiation. In addition to being implicated in organ morphogenesis, maintenance and repair, upregulated EGFR signaling has been correlated in a wide variety of tumors with progression to invasion and metastasis. Thus, EGFR and its downstream signaling molecules' are targets for therapeutic interventions in wound repair and cancer.     

Glioblastoma cells block radiation-induced programmed cell death of endothelial cells

We demonstrate that human umbilical vein endothelial cells (HUVEC) grown in co-culture (CC) with U87 glioblastoma cells transfected with green fluorescent protein (GFP-U87) exhibit resistance to radiation-mediated apoptosis. cDNA macroarray analysis reveals increases in the accumulation of RNAs for HUVEC genes encoding cell adhesion molecules, growth factor-related proteins, and cell cycle regulatory/DNA repair proteins. An increase in protein expression of integrin alphav, integrin beta1, MAPK(p42), Rad51, DNA-PK(CS), and ataxia telangiectasia gene (ATM) was detected in HUVEC grown in CC with GFP-U87 cells compared with HUVEC grown in mono-culture. Treatment with anti-VEGF antibody decreases the expression of integrin alphav, integrin beta1, DNA-PK(CS) and ATM with a corresponding increase in ionizing radiation (IR)-induced apoptosis. These data support the concept that endothelial cells growing in the tumor microenvironment may develop resistance to cytotoxic therapies due to the up-regulation by tumor cells of endothelial cells genes associated with survival.     

Comparison of cardiac stem cell sheets detached by Versene solution and from thermoresponsive dishes reveals similar properties of constructs

Cell sheets (CS) from c-kit+ cardiac stem cell (CSC) hold a potential for application in regenerative medicine. However, manufacture of CS may require thermoresponsive dishes, which increases cost and puts one in dependence on specific materials. Alternative approaches were established recently and we conducted a short study to compare approaches for detachment of CS from c-kit+ CSC. Our in-house developed method using chelation by Versene solution was compared to UpCell? thermoresponsive plates in terms of CSC proliferation, viability, gap junction formation and engraftment in a model of myocardial infarction. Use of Versene solution instead of thermoresponsive dishes resulted in comparable CS thickness (approximately 100 mcm), cell proliferation rate and no signs of apoptosis detected in both types of constructs. However, we observed a minor reduction of gap junction count in Versene-treated CS. At day 30 after delivery to infarcted myocardium both types of CS retained at the site of transplantation and contained comparable amounts of proliferating cells indicating engraftment. Thus, we may conclude that detachment of CS from c-kit+ CSC using Versene solution followed by mechanical treatment is an alternative to thermoresponsive plates allowing use of routinely available materials to generate constructs for cardiac repair.     

Simian Virus 40 Large T Antigen J Domain and Rb-Binding Motif Are Sufficient To Block Apoptosis Induced by Growth Factor Withdrawal in a Neural Stem Cell Line

Serum-free mouse embryo (SFME) cells are a neural stem cell line that is dependent upon epidermal growth factor (EGF) for survival. Removal of EGF results in the G1 arrest and apoptosis of SFME cells. We have shown that the expression of simian virus 40 large T antigen in SFME cells blocks apoptosis and allows cell survival and division in the absence of EGF. Therefore the presence of T antigen abrogates the EGF requirement. The steady-state levels of p53, p21, and mdm-2 do not increase as SFME cells undergo apoptosis upon EGF withdrawal. Furthermore, the amino-terminal 136 amino acids (N136) of T antigen are sufficient to block death and to promote proliferation in the absence of EGF, while the carboxy-terminal fragment (C251-708), which contains the p53 binding site, is unable to block death. Taken together, these data suggest that SFME cells deprived of EGF undergo p53-independent apoptosis. Mutations that disrupt either the J domain or Rb family binding abolish the ability of T antigen to block SFME cell apoptosis and to promote cell growth. We conclude that T antigen must act on one or more members of the Rb family to inhibit SFME cell apoptosis.     

Preconditioning effects of physiological cyclic stretch on pathologically mechanical stretch-induced alveolar epithelial cell apoptosis and barrier dysfunction

Background

We aim to investigate the effects of preconditioning of physiological cyclic stretch on the alveolar epithelial cell apoptosis induced by pathologically mechanical stretch and barrier dysfunction and how these effects are linked to differential expression of small GTPases Rac and Rho mRNA.

Methods

Pulmonary alveolar epithelial cells were subjected to different treatments of cyclic stretch (CS) at 5% and 20% elongation, respectively. Cells maintained in normal cell culture were used as negative control. On the other hand, cell apoptosis and Rac/Rho activities in cells with or without preconditioning of physiologically relevant magnitudes of CS (5% CS) with different durations (0, 15, 30, 60 and 120 min) in prior to 6-h treatment with pathological CS stimulation (20% CS) were compared and measured.

Results

Pathological CS could cause a significant increase in apoptosis rate, which is considered to be associated with the repression of Rac mRNA and activation of Rho mRNA. In contrast, physiological 5%-CS preconditioning suppressed cell apoptosis and induced nearly complete monolayer recovery with fewer actin stress fibers and paracellular gap formation. Consistent with differential effects on cell apoptosis and epithelial cell integrity, physiological CS preconditioning enhanced expression of Rac mRNA but inhibited Rho activation.

Conclusions

Physiological CS preconditioning has an inhibitory effect on cell apoptosis while exerts a stimulatory impact on epithelial cell recovery via regulation of Rac and Rho activities.     

The influence of epidermal growth factor on the course of ischemia-reperfusion induced pancreatitis in rats.

Acute pancreatitis is accompanied by the enhanced expression of EGF in the pancreas and the administration of EGF was found to exhibit the beneficial effect on edematous cerulein-induced pancreatitis. Therefore, we decided to determine the influence of EGF on necro-hemorrhagic pancreatitis induced by ischemia and reperfusion (I/R). Acute pancreatitis was induced in rats by restricting the pancreatic blood flow (PBF) in the inferior splenic artery for 30 min using microvascular clips. EGF was administered three times daily (10 microg/kg per dose s.c.) starting immediately after the clips removal. Rats were sacrificed on day 1, 3, 5, 10 and 21 following ischemia. PBF was measured using a laser Doppler flowmeter. Morphological signs of pancreatitis, as well as the levels of plasma amylase, lipase, interleukin-1beta and interleukin-10 concentration and pancreatic cell proliferation were examined. Results: Ischemia with reperfusion caused acute necro-hemorrhagic pancreatitis with a histological and biochemical manifestation of pancreatic damage, followed by a spontaneous regeneration. The administration of EGF caused the reduction in the histological signs of pancreatic damage, such as necrosis, edema and leukocyte infiltration, and accelerated the pancreatic repair. Also, EGF treatment significantly attenuated the reduction in pancreatic blood flow and DNA synthesis. The activity of plasma amylase and lipase, as well as plasma interleukin-1beta and interleukin-10 concentrations were decreased in EGF treated animals. CONCLUSIONS: EGF exerts beneficial influence on the course of I/R induced pancreatitis and this effect seems to be related to the reduction in the activation of pro-inflammatory interleukin cascade, the improvement of PBF, and the increase in pancreatic cell growth.     

Insulin-like growth factor I is the key growth factor in serum that protects neuroblastoma cells from hyperosmotic-induced apoptosis

Neuroblastoma is a childhood tumor of the peripheral nervous system that remains largely uncurable by conventional methods. Mannitol induces apoptosis in neuroblastoma cell types and insulin-like growth factor I (IGF-I) protects these cells from hyperosmotic-induced apoptosis by affecting apoptosis-regulatory proteins. In the current study, we investigate factors that enable SH-SY5Y neuroblastoma cells to survive in the presence of an apoptotic stimulus. When SH-SY5Y cells are exposed to high mannitol concentrations, more than 60% of the cells are apoptotic within 48 h. Normal CS prevents hyperosmotic-induced apoptosis in a dose-dependent manner, with 0.6% CS protecting 50% of the cells, and 3% CS rescuing more than 70% of the cells from apoptosis. Serum also delays the commitment point for SH-SY5Y cells from 9 h to 35 h. A survey of several growth factors, including epidermal growth factor (EGF), platelet-derived growth factor (PDGF), nerve growth factor (NGF), fibroblast growth factor (FGF), and IGF-I reveals that IGF-I is a component of serum necessary for protection of neuroblastoma cells from death. Mitochondrial membrane depolarization occurs in greater than 40% of the cells after mannitol exposure and caspase-3 activation is increased in high mannitol conditions after 9 h. IGF-I blocks both the mitochondrial membrane depolarization and caspase-3 activation normally induced by hyperosmotic treatment in neuroblastoma cells. Our results suggest that (1) IGF-I is a key factor in serum necessary for protection from death and (2) IGF-I acts upstream from the mitochondria and the caspases to prevent apoptosis in human neuroblastoma.     

Multiple requirements for SHPTP2 in epidermal growth factor-mediated cell cycle progression.

Using transient overexpression and microinjection approaches, we examined SHPTP2's function in growth factor signaling. Overexpression of catalytically inactive SHPTP2 (PTP2CS) but not catalytically inactive SHPTP1, inhibited mitogen-activated protein (MAP) kinase activation and Elk-1 transactivation following epidermal growth factor (EGF) stimulation of 293 cells. An SHPTP2 mutant with both C-terminal tyrosyl phosphorylation sites converted to phenylalanine (PTP2YF) was also without effect; moreover, PTP2YF rescued PTP2CS-induced inhibition of EGF-induced Elk-1 transactivation. PTP2CS did not inhibit transactivation by activated Ras, suggesting that SHPTP2 acts upstream of or parallel to Ras. Neither PTP2CS nor PTP2YF inhibited platelet-derived growth factor (PDGF)-induced Elk-1 transactivation. Thus, protein-tyrosine phosphatase activity, but not tyrosyl phosphorylation of SHPTP2, is required for the immediate-early responses to EGF but not to PDGF. To determine whether SHPTP2 is required later in the cell cycle, we assessed S-phase entry in NIH 3T3 cells microinjected with anti-SHPTP2 antibodies or with a glutathione S-transferase (GST) fusion protein encoding both SH2 domains (GST-SH2). Microinjection of anti-SHPTP2 antibodies prior to stimulation inhibited EGF- but no PDGF- or serum-induced S-phase entry. Anti-SHPTP2 antibodies or GST-SH2 fusion protein could inhibit EGF-induced S-phase entry for up to 8 h after EGF addition. Although MAP kinase activation was detected shortly after EGF stimulation, no MAP kinase activation was detected around the restriction point. Therefore, SHPTP2 is absolutely required for immediate-early and late events induced by some, but not all, growth factors, and the immediate-early and late signal transduction pathways regulated by SHPTP2 are distinguishable.     

EGF mediates protection against Fas-induced apoptosis by depleting and oxidizing intracellular GSH stocks

Several pieces of evidence have demonstrated the importance of reduction/oxidation (redox) signaling in biological processes, including sensitivity toward apoptosis. In parallel, it was recently reported that growth factors induce the generation of reactive oxygen species (ROS). Therefore, we tested the hypothesis that the anti-apoptotic effect of epidermal growth factor (EGF) was mediated by changes in the redox state of hepatocytes through changes in GSH stocks. Isolated mouse hepatocytes were cultured and exposed to anti-Fas stimulation in order to induce apoptosis. Cell death by apoptosis was assessed by Hoechst 33258 staining and by measuring caspase-3 proteolysis activity. Cell treatment with EGF significantly decreased total (GSx) and reduced (GSH) glutathione levels in the presence and the absence of anti-Fas. Furthermore, glutathione reductase activity was lower in EGF-treated cultures (by 28%) as compared to untreated cultures which lead to a significant decline in GSH/GSx ratio. These effects were found to be EGF-receptor tyrosine kinase activity dependent. Co-stimulation of cells with anti-Fas and EGF attenuated caspase-3 activation and cell death by apoptosis by 70%. GSH monoethylester (GSHmee) significantly attenuated the effect of EGF on GSH and GSH/GSx ratio. It caused an increase in caspase-3 activation and in the percentage of apoptotic cells in anti-Fas + EGF-treated cells, thus resulting in a 53% decline in the protective effect of EGF. In conclusion, EGF induces a significant and specific depletion and oxidization of intracellular GSH, paralleled by a protection against Fas-induced apoptosis. GSH repenishment partly counteracted these effects suggesting that GSH depletion contributed to the protective effect of EGF against caspase-3 activation and cell death by apoptosis.     

USP17 is required for peripheral trafficking of lysosomes

Expression of the deubiquitinase USP17 is induced by multiple stimuli, including cytokines (IL‐4/6), chemokines (IL‐8, SDF1), and growth factors (EGF), and several studies indicate it is required for cell proliferation and migration. However, the mechanisms via which USP17 impacts upon these cellular functions are unclear. Here, we demonstrate that USP17 depletion prevents peripheral lysosome positioning, as well as trafficking of lysosomes to the cell periphery in response to EGF stimulation. Overexpression of USP17 also increases secretion of the lysosomal protease cathepsin D. In addition, USP17 depletion impairs plasma membrane repair in cells treated with the pore‐forming toxin streptolysin O, further indicating that USP17 is required for lysosome trafficking to the plasma membrane. Finally, we demonstrate that USP17 can deubiquitinate p62, and we propose that USP17 can facilitate peripheral lysosome trafficking by opposing the E3 ligase RNF26 to untether lysosomes from the ER and facilitate lysosome peripheral trafficking, lysosome protease secretion, and plasma membrane repair.     

Phosphatidylinositol 3-kinase and protein kinase C are required for the inhibition of caspase activity by epidermal growth factor

The mechanism by which growth factors exert an anti-apoptotic function on many cell types is not well understood. This issue is addressed in relation to epidermal growth factor (EGF) which inhibits apoptosis induced by staurosporine or wortmannin in an epithelial tumour cell line (CNE-2). The presence of EGF substantially reduced the in vitro Ac-DEVD-AMC hydrolytic activity and almost completely suppressed the intracellular cleavage of poly(ADP-ribose) polymerase in staurosporine- or wortmannin-treated cells. Staurosporine but not wortmannin caused the intracellular proteolytic processing of pro-caspase-3 and this event was transiently inhibited by EGF. Staurosporine-induced apoptosis was not inhibited by EGF in the presence of wortmannin or LY294002. Similarly, EGF failed to inhibit wortmannin-induced apoptosis in the presence of staurosporine, chelerythrine chloride or G?6850. These results suggest that phosphatidylinositol 3-kinase and protein kinase C play a role in the survival function of EGF but the reduction of cellular caspase activity cannot be satisfactorily explained by a lack of pro-caspase-3 activation.