Ethylene signaling is required for the acceleration of cell death induced by the activation of AtMEK5 in Arabidopsis

Mitogen-activated protein kinases (MAPKs) are involved in the regulation of plant growth, development and responses to a wide variety of stimuli. In a conditional gain-of-function transgenic system, the activation of AtMEK5, a MAPK kinase, can in turn activate endogenous AtMAPK3 and AtMAPK6, and can lead to a striking increase in ethylene production and induce hypersensitive response (HR)-like cell death in Arabidopsis. However, the role of the increased ethylene production in regulating this HR-like cell death remains unknown. Using Arabidopsis transgenic plants that express AtMEK5(DD), an active mutant of AtMEK5 that is under the control of a steroid-inducible promoter, we tested the contribution of ethylene to cell death. We found that ethylene biosynthesis occurs before cell death. Cell death was delayed by inhibiting AtMEK5-induced ethylene production using inhibitors of ACC-synthases, ACC-oxidases or ethylene receptors. In the mutants AtMEK5(DD)/etr1-1 and AtMEK5(DD)/ein2-1, both of which showed insensitivity to ethylene, the expression of AtMEK5(DD) protein, activity of AtMAPK3 and AtMAPK6, and ethylene production were the same as those seen in AtMEK5(DD) transgenic plants, but cell death was also delayed. These data suggest that ethylene signaling perception is required to accelerate cell death that is induced by AtMEK5 activation.     

P2Y6 receptor signaling pathway mediates inflammatory responses induced by monosodium urate crystals

Gout occurs in individuals with hyperuricemia when monosodium urate (MSU) crystals precipitate in tissues and induce acute inflammation via phagocytic cells such as monocytes. MSU crystals have been demonstrated in skin diseases such as tophaceous gout or psoriasis; however, the importance of MSU crystals in the skin is totally unknown. In this study, we found that MSU crystals, through P2Y(6) receptors, stimulated normal human keratinocytes (NHK) to produce IL-1α, IL-8/CXCL8, and IL-6. P2Y(6) receptor expression increased in MSU-stimulated NHK. Both P2Y(6)-specific antagonist and P2Y(6) antisense oligonucleotides significantly inhibited the production of IL-1α, IL-8/CXCL8, and IL-6 by NHK. Similarly, the P2Y(6)-specific antagonist completely inhibited the MSU-induced production of IL-1β by THP-1 cells, a human monocytic cell line. Remarkably, the P2Y(6)-specific antagonist significantly reduced neutrophil influx in both mouse air pouch and peritonitis models. Thus, these results indicate that the P2Y(6) receptor signaling pathway may be a potential therapeutic target for MSU-associated inflammatory diseases, such as tophaceous gout.     

Probing the cell death signaling pathway of HepG2 cell line induced by copper-1,10-phenanthroline complex

Copper-1,10-phenanthroline (phen) complex [Cu(phen)₂] has been typically known as DNA-cleaving agent. And now it becomes more important for developing multifunctional drugs with its improved cytotoxic properties. In our study, we probed the cytophysiological mechanism of Cu(phen)₂. HepG2 cells were more sensitive to Cu(phen)₂ with an IC50 of 4.03 μM than other three kinds of cell lines. After treated by Cu(phen)₂, HepG2 cells had some typical morphological changes which happened to its nucleus. DNA ladder’s occurence and Annexin V-positive increased cells indicated that Cu(phen)₂ induced HepG2 cells into apoptosis. Further studies showed that Cu(phen)₂ treatment resulted in significant G₂/M phase arrest and collapse of mitochondrial membrane potential. Several cell cycle-related factors were down-regulated, including Cyclin A, Cyclin B1 and Cdc2. But p21 and p53 were up-regulated. DNA damage, microtubule disorganization and mitotic arrest through spindle assembly checkpoint activation were observed in Cu(phen)₂-treated cells. The activation of caspase-3, 8 & 9 were checked out. The increased-expression ratio of Bax/Bcl-2 was detected. The expression levels of Bcl-x_L and Bid were found to decrease. These meant that a mitochondrial-related apoptosis pathway was activated in treated HepG2 cells. Furthermore, some ER stress-associated signaling factors were found to be up-regulated, such as Grp78, XBP-1and CHOP. Ca²⁺ was also found to be released from the ER lumen. Collectively, our findings demonstrate that Cu(phen)₂ induces apoptosis in HepG2 cells via mitotic arrest and mitochondrial- and ER-stress-related signaling pathways.     

Effects of MPK3 and MPK6 kinases on the chloroplast architecture and function induced by cold acclimation in Arabidopsis

Photosynthesis Research - Exposure to low, non-freezing temperatures develops freezing tolerance in many plant species. Such process is called cold acclimation. Molecular changes undergone during...     

The essential nature of sphingolipids in plants as revealed by the functional identification and characterization of the Arabidopsis LCB1 subunit of serine palmitoyltransferase

Serine palmitoyltransferase (SPT) catalyzes the first step of sphingolipid biosynthesis. In yeast and mammalian cells, SPT is a heterodimer that consists of LCB1 and LCB2 subunits, which together form the active site of this enzyme. We show that the predicted gene for Arabidopsis thaliana LCB1 encodes a genuine subunit of SPT that rescues the sphingolipid long-chain base auxotrophy of Saccharomyces cerevisiae SPT mutants when coexpressed with Arabidopsis LCB2. In addition, homozygous T-DNA insertion mutants for At LCB1 were not recoverable, but viability was restored by complementation with the wild-type At LCB1 gene. Furthermore, partial RNA interference (RNAi) suppression of At LCB1 expression was accompanied by a marked reduction in plant size that resulted primarily from reduced cell expansion. Sphingolipid content on a weight basis was not changed significantly in the RNAi suppression plants, suggesting that plants compensate for the downregulation of sphingolipid synthesis by reduced growth. At LCB1 RNAi suppression plants also displayed altered leaf morphology and increases in relative amounts of saturated sphingolipid long-chain bases. These results demonstrate that plant SPT is a heteromeric enzyme and that sphingolipids are essential components of plant cells and contribute to growth and development.     

JNK pathway mediates apoptotic cell death induced by tumor suppressor LKB1 in Drosophila

Although recent progresses have unveiled the diverse in vivo functions of LKB1, detailed molecular mechanisms governing these processes still remain enigmatic. Here, we showed that Drosophila LKB1 negatively regulates organ growth by caspase-dependent apoptosis, without affecting cell size and cell cycle progression. Through genetic screening for LKB1 modifiers, we discovered the JNK pathway as a novel component of LKB1 signaling; the JNK pathway was activated by LKB1 and mediated the LKB1-dependent apoptosis. Consistently, LKB1-null mutant was defective in embryonic apoptosis and displayed a drastic hyperplasia in the central nervous system; these phenotypes were fully rescued by ectopic JNK activation as well as wild-type LKB1 expression. Furthermore, inhibition of LKB1 resulted in epithelial morphogenesis failure, which was associated with a decrease in JNK activity. Collectively, our studies unprecedentedly elucidate JNK as the downstream mediator of the LKB1-dependent apoptosis, and provide a new paradigm for understanding the diverse LKB1 functions in vivo.     

Chloroplasts of Arabidopsis are the source and a primary target of a plant-specific programmed cell death signaling pathway

Enhanced levels of singlet oxygen ((1)O(2)) in chloroplasts trigger programmed cell death. The impact of (1)O(2) production in chloroplasts was monitored first in the conditional fluorescent (flu) mutant of Arabidopsis thaliana that accumulates (1)O(2) upon a dark/light shift. The onset of (1)O(2) production is rapidly followed by a loss of chloroplast integrity that precedes the rupture of the central vacuole and the final collapse of the cell. Inactivation of the two plastid proteins EXECUTER (EX1) and EX2 in the flu mutant abrogates these responses, indicating that disintegration of chloroplasts is due to EX-dependent signaling rather than (1)O(2) directly. In flu seedlings, (1)O(2)-mediated cell death signaling operates as a default pathway that results in seedlings committing suicide. By contrast, EX-dependent signaling in the wild type induces the formation of microlesions without decreasing the viability of seedlings. (1)O(2)-mediated and EX-dependent loss of plastid integrity and cell death in these plants occurs only in cells containing fully developed chloroplasts. Our findings support an as yet unreported signaling role of (1)O(2) in the wild type exposed to mild light stress that invokes photoinhibition of photosystem II without causing photooxidative damage of the plant.     

The disease resistance signaling components EDS1 and PAD4 are essential regulators of the cell death pathway controlled by LSD1 in Arabidopsis

Specific recognition of pathogens is mediated by plant disease resistance (R) genes and translated into a successful defense response. The extent of associated hypersensitive cell death varies from none to an area encompassing cells surrounding an infection site, depending on the R gene activated. We constructed double mutants in Arabidopsis between positive regulators of R function and a negative regulator of cell death, LSD1, to address whether genes required for normal R function also regulate the runaway cell death observed in lsd1 mutants. We report here that EDS1 and PAD4, two signaling genes that mediate some but not all R responses, also are required for runaway cell death in the lsd1 mutant. Importantly, this novel function of EDS1 and PAD4 is operative when runaway cell death in lsd1 is initiated through an R gene that does not require EDS1 or PAD4 for disease resistance. NDR1, another component of R signaling, also contributes to the control of plant cell death. The roles of EDS1 and PAD4 in regulating lsd1 runaway cell death are related to the interpretation of reactive oxygen intermediate-derived signals at infection sites. We further demonstrate that the fate of superoxide at infection sites is different from that observed at the leading margins of runaway cell death lesions in lsd1 mutants.     

Intracellular signaling pathways involved in post-mitotic dopaminergic PC12 cell death induced by 6-hydroxydopamine

Oxidative stress has been shown to mediate neuron damage in Parkinson's disease (PD). In the present report, we intend to clarify the intracellular pathways mediating dopaminergic neuron death after oxidative stress production using post-mitotic PC12 cells treated with the neurotoxin 6-hydroxydopamine (6-OHDA). The use of post-mitotic cells is crucial, because one of the suggested intracellular pathways implicated in neuron death relates to the re-entry of neurons (post-mitotic cells) in the cell cycle. We find that 6-OHDA sequentially increases intracellular oxidants, functional cell damage and caspase-3 activation, leading to cell death after 12 h of incubation. Prevention of cell damage by different antioxidants supports the implication of oxidative stress in the observed neurotoxicity. Oxidative stress-dependent phosphorylation of the MAPK JNK and oxidative stress-independent PKB/Akt dephosphorylation are involved in 6-OHDA neurotoxicity. Decrease in p21(WAF1/CIP1) and cyclin-D1 expression, disappearance of the non-phosphorylated band of retinoblastoma protein (pRb), and expression of proliferating cell nuclear antigen, not present in PC12 post-mitotic cells, suggest a re-entry of differentiated cells into cell cycle. Our results indicate that such a re-entry is mediated by oxidative stress and is involved in 6-OHDA-induced cell death. We conclude that at least three intracellular pathways are involved in 6-OHDA-induced cell death in differentiated PC12 cells: JNK activation, cell cycle progression (both oxidative stress-dependent), and Akt dephosphorylation (not related to the increase of oxidants); the three pathways are necessary for the cells to die, since blocking one of them is sufficient to keep the cells alive.     

A signaling pathway, independent of the oxidative burst, that leads to hypersensitive cell death in cultured tobacco cells includes a serine protease

To examine the role of reactive oxygen species (ROS) in the signal transduction that leads to hypersensitive cell death, we used a previously established system in which a xylanase from Trichoderma viride (TvX) induces an oxidative burst and cell death in a culture of tobacco cells. Diphenylene iodonium and N-Acetyl-L-cysteine known as an inhibitor of NADPH oxidase and a scavenger of superoxides, respectively, and catalase inhibited the oxidative burst but did not inhibit the induction of cell death. We also found that inhibitors of serine proteases inhibited TvX-induced cell death. These results suggest that there is a signaling pathway in which a serine protease might be responsible for the signal transduction, which is independent of the oxidative burst, that leads to the hypersensitive cell death of tobacco cells.     

Metacaspase-8 modulates programmed cell death induced by ultraviolet light and H2O2 in Arabidopsis

Programmed cell death (PCD) is a genetically controlled cell death that is regulated during development and activated in response to environmental stresses or pathogen infection. The degree of conservation of PCD across kingdoms and phylum is not yet clear; however, whereas caspases are proteases that act as key components of animal apoptosis, plants have no orthologous caspase sequences in their genomes. The discovery of plant and fungi metacaspases as proteases most closely related to animal caspases led to the hypothesis that metacaspases are the functional homologues of animal caspases in these organisms. Arabidopsis thaliana has nine metacaspase genes, and so far it is unknown which members of the family if any are involved in the regulation of PCD. We show here that metacaspase-8 (AtMC8) is a member of the gene family strongly up-regulated by oxidative stresses caused by UVC, H(2)O(2), or methyl viologen. This up-regulation was dependent of RCD1, a mediator of the oxidative stress response. Recombinant metacaspase-8 cleaved after arginine, had a pH optimum of 8, and complemented the H(2)O(2) no-death phenotype of a yeast metacaspase knock-out. Overexpressing AtMC8 up-regulated PCD induced by UVC or H(2)O(2), and knocking out AtMC8 reduced cell death triggered by UVC and H(2)O(2) in protoplasts. Knock-out seeds and seedlings had an increased tolerance to the herbicide methyl viologen. We suggest that metacaspase-8 is part of an evolutionary conserved PCD pathway activated by oxidative stress.     

The gain-of-function Arabidopsis acd6 mutant reveals novel regulation and function of the salicylic acid signaling pathway in controlling cell death, defenses, and cell growth.

We isolated a dominant gain-of-function Arabidopsis mutant, accelerated cell death 6 (acd6), with elevated defenses, patches of dead and enlarged cells, reduced stature, and increased resistance to Pseudomonas syringae. The acd6-conferred phenotypes are suppressed by removing a key signaling molecule, salicylic acid (SA), by using the nahG transgene, which encodes SA hydroxylase. This suppression includes phenotypes that are not induced by application of SA to wild-type plants, indicating that SA acts with a second signal to cause many acd6-conferred phenotypes. acd6-nahG plants show hyperactivation of all acd6-conferred phenotypes after treatment with a synthetic inducer of the SA pathway, benzo(1,2, 3)thiadiazole-7-carbothioic acid (BTH), suggesting that SA acts with and also modulates the levels and/or activity of the second defense signal. acd6 acts partially through a NONEXPRESSOR OF PR 1 (NPR1) gene-independent pathway that activates defenses and confers resistance to P. syringae. Surprisingly, BTH-treated acd6-nahG plants develop many tumor-like abnormal growths, indicating a possible role for SA in modulating cell growth.     

The ARG1-LIKE2 gene of Arabidopsis functions in a gravity signal transduction pathway that is genetically distinct from the PGM pathway

The arl2 mutants of Arabidopsis display altered root and hypocotyl gravitropism, whereas their inflorescence stems are fully gravitropic. Interestingly, mutant roots respond like the wild type to phytohormones and an inhibitor of polar auxin transport. Also, their cap columella cells accumulate starch similarly to wild-type cells, and mutant hypocotyls display strong phototropic responses to lateral light stimulation. The ARL2 gene encodes a DnaJ-like protein similar to ARG1, another protein previously implicated in gravity signal transduction in Arabidopsis seedlings. ARL2 is expressed at low levels in all organs of seedlings and plants. arl2-1 arg1-2 double mutant roots display kinetics of gravitropism similar to those of single mutants. However, double mutants carrying both arl2-1 and pgm-1 (a mutation in the starch-biosynthetic gene PHOSPHOGLUCOMUTASE) at the homozygous state display a more pronounced root gravitropic defect than the single mutants. On the other hand, seedlings with a null mutation in ARL1, a paralog of ARG1 and ARL2, behave similarly to the wild type in gravitropism and other related assays. Taken together, the results suggest that ARG1 and ARL2 function in the same gravity signal transduction pathway in the hypocotyl and root of Arabidopsis seedlings, distinct from the pathway involving PGM.     

Characterization of the cell death induced by cadmium in HaCaT and C6 cell lines

Cell death resulting from cadmium (Cd) intoxication has been confirmed to induce both necrosis and apoptosis. The ratio between both types of cell death is dose- and cell-type-dependent. This study used the human keratinocytes HaCaT expressing a mutated p53 and the rat glial cells C6 expressing a wild p53 as models to characterize Cd-induced apoptosis, using sub-lethal and lethal doses. At these concentrations, features of apoptosis were observed 24 h after C6 cell treatment: apoptotic DNA fragmentation and caspase-9 activation, whereas Cd did not induce caspase-3. In HaCaT, Cd did not induce apoptotic DNA fragmentation or caspase-9 and -3 activation. The results also showed that the inhibition of p53 led to a resistance of the C6 cells to 20 µm Cd, decreased the apoptosis and increased the metallothioneins in these cells. p53 restoration increased the sensitivity of HaCaT cells to Cd but did not affect the MT expression. The results suggest that Cd induced apoptosis in C6 cells but a non-apoptotic cellular death in HaCaT cells.