Endoplasmic reticulum stress-induced cell death mediated by the proteasome

Cells exposed to sustained endoplasmic reticulum (ER) stress undergo programmed cell death and display features typical of apoptosis, such as cysteine aspartyl protease (caspase) activation, cytochrome c release, and DNA fragmentation. Here, we show that the execution of cell death induced by ER stress is mediated via the proteasome. Inhibition of the proteasome by lactacystin prevented ER stress-induced degradation of Bcl-2, release of cytochrome c, processing of effector caspase-3, and exposure of phosphatidylserine. Owing to the ability of lactacystin to inhibit cytochrome c release, we propose that the pro-apoptotic activity of the proteasome lies upstream of mitochondrial activation. Thus, the proteasome serves as a principal mediator of ER stress-induced cell death in this system.     

泛素/26S蛋白酶体系统介导的细胞程序化死亡

在一定的生理或者病理条件下,细胞为了自身发育或者抵御不良刺激,会采取细胞程序化死亡（programmed cell death,PCD）的方式结束生命。泛素/26S蛋白酶体系统（ubiquitin-26S proteasome system,UPS）作为生物体中重要的翻译后蛋白质调节系统,对PCD起着关键的调节作用。该文介绍UPS通过两条细胞凋亡信号转导通路以及天冬氨酸特异性半胱氨酸蛋白酶来调控PCD的研究进展。     

Mammary epithelial reorganization on extracellular matrix is mediated by cell surface galactosyltransferase.

When plated at appropriate densities in serum-free media, the COMMA-D mammary epithelial cell line rapidly reorganizes into multicellular spheres on the basement membrane matrix derived from Engelbreth-Holm-Swarm murine tumor. Using time-lapse video-microscopy, four stages of reorganization were discerned during the first 24 h of culture. In the first few hours, cells attached to the matrix, elongated, migrated, and formed chains. In the next 6 h, chains of cells linked together in anastomosing networks. In the period between 8 and 18 h postplating, the networks contracted, resulting in dense cords radiating from central aggregates. During the final 6 h, the cords were drawn into the aggregates, which condensed further into spheres. The events occurring during mammary epithelial cell reorganization on the matrix were shown to be mediated by cell surface beta-1,4-galactosyltransferase (GalTase), a receptor that binds N-acetylglucosamine residues on glycosylated proteins. GalTase activity was evident at the surface of cells cultured on reconstituted matrix for 3 h but was absent from cells on glass. The protein alpha-lactalbumin (alpha-LA) inhibits the association of GalTase with N-acetylglucosamine. alpha-LA present from the beginning of culture on reconstituted matrix had no effect on cell attachment but caused concentration-dependent inhibition of the first two steps of reorganization, i.e., cell elongation and network formation, which then interfered with subsequent events. These observations were replicated using polyclonal antibodies to GalTase. Reorganization was impaired when alpha-LA was added during the first two stages but no effect was observed when it was added during the last two stages. Cells cultured on plastic, which lack surface GalTase activity, were unperturbed by incubation with alpha-LA. Thus certain events (cell elongation and network elaboration) during mammary epithelial cell reorganization on reconstituted matrix are GalTase dependent, while others (attachment, network contraction, and compaction) are not. The functional and temporal specificity of GalTase involvement indicates that GalTase mediates cell-matrix, but not cell-cell, interactions during epithelial morphogenetic events in culture.     

Regulation of Snf1 kinase. Activation requires phosphorylation of threonine 210 by an upstream kinase as well as a distinct step mediated by the Snf4 subunit

The yeast Snf1 kinase and its metazoan orthologues, the AMP-activated protein kinases, are activated in response to nutrient limitation. Activation requires the phosphorylation of a conserved threonine residue in the activation loop of the catalytic subunit. A phosphopeptide antibody was generated that specifically recognizes Snf1 protein that is phosphorylated in its activation loop on threonine 210. Using this reagent, we show that phosphorylation of threonine 210 correlates with Snf1 activity, since it is detected in cells subjected to glucose limitation but not in cells grown in abundant glucose. A Snf1 mutant completely lacking kinase activity was phosphorylated normally on threonine 210 in glucose-starved cells, eliminating the possibility that the threonine 210 modification is due to an autophosphorylation event. Cells lacking the Reg1 protein, a regulatory subunit for the Glc7 phosphatase, showed constitutive phosphorylation of Snf1 threonine 210. Exposure of cells to high concentrations of sodium chloride also induced phosphorylation of Snf1. Interestingly, Mig1, a downstream target of Snf1 kinase, is phosphorylated in glucose-stressed but not sodium-stressed cells. Finally, cells lacking the gamma subunit of the Snf1 kinase complex encoded by the SNF4 gene exhibited normal regulation of threonine 210 phosphorylation in response to glucose limitation but are unable to phosphorylate Mig1 efficiently. Our data indicate that activation of the Snf1 kinase complex involves two steps, one that requires a distinct upstream kinase and one that is mediated by the gamma subunit of the kinase itself.     

DNA synthesis by CHO cell extracts on fork-like DNA templates containing the major cisplatin adduct requires a ligation step

In this work we have examined the role of DNA ligation in the in vitro replication catalyzed by CHO crude extracts on fork-like oligonucleotide substrates containing a unique d(GpG) intrastrand cross-link produced by the antitumor drug cisplatin. We show here that this reaction involves a ligation step, which necessitates excision of the flap strand of the forked substrate. By constructing substrates in which the unannealed tail could not be degraded by a 5' exonuclease, we obtained evidence suggesting that this type of activity participates in the removal of the flap strand. Furthermore, we found that the ligation event played a predominant role in the synthesis of fully replicated products from both intact and platinated templates. Finally, we investigated whether translesion synthesis of the cisplatin lesion could occur concomitantly to ligation by monitoring the incorporation of labeled precursors downstream of the adduct. Our results are compatible with the possibility that some translesion syntheses of the Pt-d(GpG) adduct by the extracts also contributed to the generation of full length molecules.     

Cell-cell contacts in the human cell line ECV304 exhibit both endothelial and epithelial characteristics

Endothelial cells separate the intra- and extravascular space and regulate transport processes between these compartments. Since intercellular junctions are required for these specific cell functions, the cell-cell contacts in the permanent cell line ECV304 were systematically analyzed and compared with human umbilical vein endothelial cells (HUVECs) in primary culture and with the epithelial Madin Darby Canine Kidney (MDCK) cell line. Filter-grown ECV304 cells generate a distinct electrical resistance and a permeability barrier between cell culture compartments. Electron microscopy of ECV304 cells revealed lateral membrane interdigitations, typically found in endothelial cells in vivo, with direct membrane contact sites, which prevented the diffusion of lanthanum. By immunoblot and immunofluorescence analysis, the expression and cellular localization of the tight junction and adherens-type junction proteins occludin, ZO-1, symplekin, beta-catenin, and plakoglobin were analyzed. ECV304 cells display further characteristics of endothelial cells, including the expresssion of thrombomodulin and of the vitronectin receptor CD51, as well as the secretion of plasminogen activator inhibitor 1 (PAI-1) and endothelin. However, ECV304 cells also express proteins characteristically found in epithelial cells, including E-cadherin and the desmosomal proteins desmoplakin, desmocollin, and desmoglein; occasionally desmosomal structures can be identified by electron microscopy. In conclusion, ECV304 cells express many endothelial markers and form specialized intercellular junctions that display some epithelial features. Thus this reportedly endothelial-derived permanent human cell line may be dedifferentiated toward an epithelial phenotype.     

Evidence for proteasome dysfunction in cytotoxicity mediated by anti-Ras intracellular antibodies.

Anti-Ras intracellular antibodies inhibit cell proliferation in vivo by sequestering the antigen and diverting it from its physiological location [Lener, M., Horn, I. R., Cardinale, A., Messina, S., Nielsen, U.B., Rybak, S.M., Hoogenboom, H.R., Cattaneo, A., Biocca, S. (2000) Eur. J. Biochem.267, 1196-1205]. Here we demonstrate that strongly aggregating single-chain antibody fragments (scFv), binding to Ras, induce apoptosis, and this effect is strictly related to the antibody-mediated aggregation of p21Ras. Proteasomes are quickly recruited to the newly formed aggregates, and their activity is strongly inhibited. This leads to the formation of aggresome-like structures, which become evident in the vast majority of apoptotic cells. A combination of anti-Ras scFv fragments with a nontoxic concentration of the proteasome inhibitor, lactacystin, markedly increases proteasome dysfunction and apoptosis. The dominant-negative H-ras (N17-H-ras), which is mostly soluble and does not induce aggresome formation or inhibit proteasome activity, only affects cell viability slightly. Together, these observations suggest a mechanism linking antibody-mediated Ras aggregation, impairment of the ubiquitin-proteasome system, and cytotoxicity.     

The degradation of nascent fibrinogen chains is mediated by the ubiquitin proteasome pathway.

Recent studies have shown that ubiquitin-dependent proteolysis by proteasomes plays an essential role in the degradation of ER-retained proteins. We investigated the degradation of individual fibrinogen chains in transfected COS cells which express but do not secrete single chains. In transfected COS cells, the degradation of fibrinogen Bbeta and gamma chain was markedly inhibited by the proteasome inhibitors lactacystin and MG132. These specific proteasome inhibitors also partially affected the degradation of Aalpha chain. In HepG2 cells, which synthesize and secrete fibrinogen, the degradation of intracellular free gamma chain was also inhibited by MG132. We also detected high molecular weight polyubiquitinated forms of fibrinogen chains in transfected COS cells and in HepG2 cells by sequential immunoprecipitation. These results implicate proteasomes in the degradation of fibrinogen chains. In COS cells, gamma chains have a longer half-life than Bbeta chains and Aalpha chains, suggesting that the presence of surplus gamma chains in fibrinogen-producing cells is due to the unequal degradation rate of fibrinogen chains. These results indicate that the ubiquitin-proteasome pathway may be a major system for the degradation of unassembled fibrinogen chains.     

Regulation of cell adhesion by protein-tyrosine phosphatases: II. Cell-cell adhesion

Cell-cell adhesion is critical to the development and maintenance of multicellular organisms. The stability of many adhesions is regulated by protein tyrosine phosphorylation of cell adhesion molecules and their associated components, with high levels of phosphorylation promoting disassembly. The level of tyrosine phosphorylation reflects the balance between protein-tyrosine kinase and protein-tyrosine phosphatase activity. Many protein-tyrosine phosphatases associate with the cadherin-catenin complex, directly regulating the phosphorylation of these proteins, thereby affecting their interactions and the integrity of cell-cell junctions. Tyrosine phosphatases can also affect cell-cell adhesions indirectly by regulating the signaling pathways that control the activities of Rho family G proteins. In addition, receptor-type tyrosine phosphatases can mediate outside-in signaling through both ligand binding and dimerization of their extracellular domains. This review will discuss the role of protein-tyrosine phosphatases in cell-cell interactions, with an emphasis on cadherin-mediated adhesions.     

Drosophila primordial germ cell migration requires epithelial remodeling of the endoderm

Trans-epithelial migration describes the ability of migrating cells to cross epithelial tissues and occurs during development, infection, inflammation, immune surveillance, wound healing and cancer metastasis. Here we investigate Drosophila primordial germ cells (PGCs), which migrate through the endodermal epithelium. Through live imaging and genetic experimentation we demonstrate that PGCs take advantage of endodermal tissue remodeling to gain access to the gonadal mesoderm and are unable to migrate through intact epithelial tissues. These results are in contrast to the behavior of leukocytes, which actively loosen epithelial junctions to migrate, and raise the possibility that in other contexts in which migrating cells appear to breach tissue barriers, they are actually exploiting existing tissue permeability. Therefore, the use of active invasive programs is not the sole mechanism to infiltrate tissues.     

Biophysical forces mediated by respiration maintain lung alveolar epithelial cell fate

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Expression of epithelial cell iron-related genes upon infection by Neisseria meningitidis

Infection by the obligate human pathogens Neisseria meningitidis (MC) and Neisseria gonorrhoeae (GC) reduces the expression of host epithelial cell transferrin receptor 1 (TfR-1) (Bonnah et al., 2000, Cellular Microbiology 2: 207-218). In addition, the rate and pattern of TfR-1 cycling is altered, leading to diminished uptake of Tf-iron by infected host cells. As Tf-iron is important for maintaining iron homeostasis in the eukaryotic cell, these findings raised the possibility that Neisseria infection might affect further pathways of epithelial cell iron metabolism. We used a specialized cDNA microarray platform, the 'IronChip', to investigate the expression of genes involved in iron transport, storage and regulation. We show that mRNA expression of several host genes involved in iron homeostasis is altered. Surprisingly, the general mRNA expression profile of infected cells closely resembled that of uninfected cells grown in an iron-limited environment. An important exception to this profile is TfR-1, the mRNA level of which is strongly reduced. Low TfR-1 expression may be explained in part by decreased activity of the iron-regulatory proteins (IRPs) in MC-infected cells, which may result in the destabilization of TfR-1 mRNA. Intriguingly, low IRP activity contrasts with the decrease in H-ferritin protein levels in infected cells. This finding suggests that low IRP activity may be responsible in part for the decrease in TfR-1 mRNA levels. A discussion of these novel findings in relation to MC infection and virulence is provided.     

Extracellular ATP facilitates cell extrusion from epithelial layers mediated by cell competition or apoptosis

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TGF-beta induced G(1) cell cycle arrest requires the activity of the proteasome pathway. Transforming growth factor

Transforming growth factor-beta (TGF-beta) induces a potent G(1)/S-phase cell cycle arrest of epithelial cells by inhibiting the activities of cyclin D- and cyclin E-associated kinase complexes. Downregulation of the kinase activities is mediated by induction of cyclin dependent kinase (CDK) inhibitor p15(Ink4b) which blocks CDK4 and CDK6 kinases and leads to binding of p27(Kip1) to CDK2-cyclin E complex. Levels of several of these factors are controlled by the ubiquitin-proteasome pathway. We demonstrate here that proteasomal inhibitors release the cells from TGF-beta imposed G(1)-phase arrest and instigate the entry of the cells into S-phase. Proteasomal inhibitors are shown to specifically increase the activity of the cyclin D-kinase complex by increasing the levels of p27(Kip1) and cyclin D and by maintaining CDK4/6 protein levels leading to phosphorylation of the retinoblastoma protein without increasing cyclin E-associated kinase activity. The results indicate caution in the potential therapeutic use of the proteasome inhibitors due to unscheduled initiation of DNA replication in the presence of a physiological growth inhibitor.     

Oxidative stress-induced intestinal epithelial cell apoptosis is mediated by p38 MAPK

Free oxygen radicals are involved in the pathogenesis of necrotizing enterocolitis (NEC) in premature infants. The stress-activated p38 mitogen-activated protein kinase (MAPK) has been implicated in gut injury. Here, we found that phosphorylated p38 was detected primarily in the villus tips of normal intestine, whereas it was expressed in the entire mucosa in NEC. H(2)O(2) treatment resulted in a rapid phosphorylation of p38 MAPK and subsequent apoptosis of rat intestinal epithelial (RIE)-1 cells; this induction was attenuated by treatment with SB203580, a selective p38 MAPK inhibitor, or transfection with p38alpha siRNA. Moreover, SB203580 also blocked H(2)O(2)-induced PKC activation. In contrast, the PKC inhibitor (GF109203x) did not affect p38 activation, indicating that p38 MAPK activation occurs upstream of PKC activation in H(2)O(2)-induced apoptosis. H(2)O(2) treatment also decreased mitochondrial membrane potential; pretreatment with SB203580 attenuated this response. Our study demonstrates that the p38 MAPK/PKC pathway plays an important role as a pro-apoptotic cellular signaling during oxidative stress-induced intestinal epithelial cell injury.     

Intestinal epithelial cell dysfunction is mediated by an endothelial-specific radiation-induced bystander effect

The response of endothelial cells (EC) to high radiation doses leads to damage of normal tissue or tumor. The precise mechanisms of the endothelial-tissue linkage are still largely unknown. We investigated the possible involvement of a bystander effect, secondary to endothelial damage, in tissue response to radiation. Proliferating human intestinal epithelial T84 cells were grown in a non-contact co-culture with confluent primary human microvascular EC (HMVEC-L). The bystander response in unirradiated T84 cells co-cultured with irradiated EC was studied by evaluating cell growth, cell death and epithelial morphology. Twenty-four hours after exposure of EC to 15 Gy, unirradiated T84 cells showed a decreased cell number (29%) and percentage in mitosis (66%) as well as increased apoptosis (1.5-fold) and cell surface area (1.5-fold), highlighting the involvement of bystander effects on T84 cells after irradiation of EC. Furthermore, the responses of T84 cells were amplified when EC and T84 cells were irradiated together, indicating that the bystander response in T84 cells adds further to direct radiation damage. As opposed to direct irradiation, the T84 cell bystander response did not involve the cell cycle-related protein p21(Waf1) (CDKN1A) and pro-apoptosis protein BAX. The bystander effect was specific to EC since the irradiation of human colon fibroblasts did not induce bystander responses in unirradiated T84 cells. These results strengthen previous in vivo evidence of the role of EC in tissue damage by radiation. In addition, this study provides a suitable and useful model to identify soluble factors involved in bystander effects secondary to endothelial damage. Modulating such factors may have important clinical implications.     

Carbon nanoparticle-induced lung epithelial cell proliferation is mediated by receptor-dependent Akt activation

Treatment of lung epithelial cells with different kinds of nano-sized particles leads to cell proliferation. Because bigger particles fail to induce this reaction, it is suggested that the special surface properties, due to the extremely small size of these kinds of materials, is the common principle responsible for this specific cell reaction. Here the activation of the protein kinase B (Akt) signaling cascade by carbon nanoparticles was investigated with regard to its relevance for proliferation. Kinetics and dose-response experiments demonstrated that Akt is specifically activated by nanoparticulate carbon particles in rat alveolar type II epithelial cells as well as in human bronchial epithelial cells. This pathway appeared to be dependent on epidermal growth factor receptor and beta(1)-integrins. The activation of Akt by these receptors is known to be a feature of adhesion-dependent signaling. However, intracellular proteins described in this context (focal adhesion kinase pp125(FAK) and integrin-linked kinase) were not activated, indicating a specific signaling mechanism. Inhibitor studies demonstrate that nanoparticle-induced proliferation is mediated by phosphoinositide 3-kinases and Akt. Moreover, overexpression of mutant Akt, as well as pretreatment with an Akt inhibitor, reduced nanoparticle-specific ERK1/2 phosphorylation, which is decisive for nanoparticle-induced proliferation. With this report, we describe the activation of a pathway by carbon nanoparticles that was so far known to be triggered by ligand receptor binding or on cell adhesion to extracellular matrix proteins.