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1.
J Plant Physiol ; 171(3-4): 276-84, 2014 Feb 15.
Article in English | MEDLINE | ID: mdl-24120533

ABSTRACT

The presence and activation of MAPK-like proteins in intertidal macroalgae is described in the current study. Two MAPK-like proteins of 40 and 42 kDa in size similar to p38 and JNK, of mammalian cells have been identified in six representative species of intertidal macroalgae from the Strait of Gibraltar (Southern Spain), namely in the chlorophytes Ulva rigida and Chaetomorpha aerea, the rhodophytes Corallina elongata and Jania rubens, and the phaeophytes Dictyota dichotoma and Dilophus spiralis. Phosphorylation of MAPK-like proteins was studied during semi-tidal cycles. Analysis of p38-like and JNK-like MAPKs in macroalgae protein extracts was carried out by using specific antibodies against the phosphorylated forms of both MAPKs. Protein blot analysis of samples collected from 2009 to 2011 in natural growing sites on days when either low or high tide occurred at midday, indicated that MAPK-like proteins in all species were highly phosphorylated in response to desiccation imposed by low tide or high irradiance. Phosphorylation of p38-like MAPK always preceded that of JNK-like MAPK. In addition, phosphorylation of MAPKs was fastest in rhodophytes, followed by chlorophytes and then finally phaeophytes. In the first group, phosphorylation was mostly dependent on desiccation, whereas both high irradiance and desiccation were responsible for p38-like and JNK-like phosphorylation in chlorophytes. In phaeophytes, high irradiance was mostly responsible for MAPK-like activation.


Subject(s)
Environment , Mitogen-Activated Protein Kinases/metabolism , Plant Proteins/metabolism , Seaweed/enzymology , Stress, Physiological , Mitogen-Activated Protein Kinases/genetics , Phosphorylation , Plant Proteins/genetics , Seaweed/genetics
2.
J Exp Bot ; 60(3): 815-28, 2009.
Article in English | MEDLINE | ID: mdl-19251986

ABSTRACT

Programmed cell death is necessary for homeostasis in multicellular organisms and it is also widely recognized to occur in unicellular organisms. However, the mechanisms through which it occurs in unicells, and the enzymes involved within the final response is still the subject of heated debate. It is shown here that exposure of the unicellular microalga Dunaliella viridis to several environmental stresses, induced different cell death morphotypes, depending on the stimulus received. Senescent cells demonstrated classical and unambiguous apoptotic-like characteristics such as chromatin condensation, DNA fragmentation, intact organelles, and blebbing of the cell membrane. Acute heat shock caused general swelling and altered plasma membrane, but the presence of chromatin clusters and DNA strand breaks suggested a necrotic-like event. UV irradiated cells presented changes typical for necrosis, together with apoptotic characteristics resembling an intermediate cell-death phenotype termed aponecrosis-like. Cells subjected to hyperosmotic shock revealed chromatin spotting without DNA fragmentation, and extensive cytoplasmic swelling and vacuolization, comparable to a paraptotic-like cell death phenotype. Nitrogen-starved cells showed pyknosis, blebbing, and cytoplasmic consumption, indicating a similarity to autophagic/vacuolar-like cell death. The caspase-like activity DEVDase was measured by using the fluorescent substrate Ac-DEVD-AMC and antibodies against the human caspase-3 active enzyme cross-reacted with bands, the intensity of which paralleled the activity. All the environmental stresses tested produced a substantial increase in both DEVDase activity and protein levels. The irreversible caspase-3 inhibitor Z-DEVD-FMK completely inhibited the enzymatic activity whereas serine and aspartyl proteases inhibitors did not. These results show that cell death in D. viridis does not conform to a single pattern and that environmental stimuli may produce different types of cell death depending on the type and intensity of the stimulus, all of which help to understand the cell death-dependent and cell death-independent functions of caspase-like proteins. Hence, these data support the theory that alternative, non-apoptotic programmed cell death (PCDs), exist either in parallel or in an independent manner with apoptosis and were already present in single-celled organisms that evolved some 1.2-1.6 billion years ago.


Subject(s)
Caspases/metabolism , Chlorophyta/cytology , Chlorophyta/enzymology , Environment , Peptide Hydrolases/metabolism , Stress, Physiological , Cell Death , Cell Shape , Chlorophyta/ultrastructure , DNA Breaks , DNA Fragmentation , DNA, Plant/metabolism , Immunoblotting , Plant Proteins/metabolism
3.
Nephron Physiol ; 109(1): p1-10, 2008.
Article in English | MEDLINE | ID: mdl-18460876

ABSTRACT

BACKGROUND: The genomic response to adaptation of IMCD3 cells to hypertonicity results in both upregulation and downregulation of a variety of genes. METHOD: The present study was undertaken to assess the metabonomic and proteomic response of IMCD3 cells that have been chronically adapted to hypertonicity (600 and 900 mosm/kg H(2)O) as compared to cells under isotonic conditions. RESULTS: Adaptation of IMCD3 cells to hypertonic conditions resulted in a change of a wide range of organic osmolytes, including sorbitol (+8,291%), betaine (+1,099%), myo-inositol (+669%), taurine (+113%) and glycerophosphorylcholine (+61%). Evaluation of the polyol pathway for sorbitol production revealed a reduction in sorbitol dehydrogenase and an increase in aldose reductase mRNA in adapted cells. Proteome analysis revealed increased expression of six glycolytic proteins, including malic enzyme and pyruvate carboxylase, indicating the activation of the pyruvate shunt and changes in glucose metabolism. This study showed that the observed reduction in cell replication could possibly reflect a redirection of cellular energy from cell growth and replication to maintenance of intracellular ion levels in chronically adapted cells. CONCLUSION: The combined metabonomic and proteomic analysis was shown to be a very helpful tool for the analysis of the effects caused by chronic adaptation to hypertonicity. It made it possible to better evaluate the importance of certain changes that occur in the process of adaptation.


Subject(s)
Energy Metabolism , Enzymes/metabolism , Kidney Medulla/metabolism , Kidney Tubules, Collecting/metabolism , Proteomics , Adaptation, Physiological , Amino Acids/metabolism , Animals , Blotting, Western , Cell Line , Cell Proliferation , Electrophoresis, Gel, Two-Dimensional , Enzymes/genetics , Glucose/metabolism , Kidney Medulla/enzymology , Kidney Medulla/ultrastructure , Kidney Tubules, Collecting/enzymology , Kidney Tubules, Collecting/ultrastructure , Mice , Microscopy, Electron, Transmission , Mitochondria/metabolism , Nuclear Magnetic Resonance, Biomolecular , Osmotic Pressure , Phenotype , Phosphates/metabolism , Polymers/metabolism , Proteomics/methods , Reverse Transcriptase Polymerase Chain Reaction , Saline Solution, Hypertonic
4.
Proc Natl Acad Sci U S A ; 104(34): 13672-7, 2007 Aug 21.
Article in English | MEDLINE | ID: mdl-17690246

ABSTRACT

Antibody array proteomics was used to detect differentially expressed proteins in inner medullary collecting duct 3 (IMCD3) cells grown under isotonic and chronic hypertonic conditions. Of 512 potential proteins, >90% were unchanged in expression. Noteworthy was the up-regulation of several tight junction-related proteins, including MUPP1 (multi-PDZ protein-1), ZO1 (zonula occludens 1), and Af6. The most robustly up-regulated protein under hypertonic conditions was MUPP1 (7.2x, P < 0.001). Changes in expression for MUPP1 were verified by quantitative PCR for message and Western blot for protein. In mouse kidney tissues, MUPP1 expression was substantial in the papilla and was absent in the cortex. Furthermore, MUPP1 expression increased 253% (P < 0.01) in the papilla upon 36 h of thirsting. Localization of MUPP1 protein expression was confirmed by immunocytochemical analysis demonstrating only minor staining under isotonic conditions and the substantial presence in chronically adapted cells at the basolateral membrane. Message and protein half-life in IMCD3 cells were 26.2 and 17.8 h, respectively. Osmotic initiators of MUPP1 expression included NaCl, sucrose, mannitol, sodium acetate, and choline chloride but not urea. Stable IMCD3 clones silenced for MUPP1 expression used the pSM2-MUPP1 vector. In cell viability experiments, clones silenced for MUPP1 demonstrated only a minor loss in cell survival under acute sublethal osmotic stress compared with empty vector control cells. In contrast, a 24% loss (P < 0.02) in transepithelial resistance for monolayers of MUPP1-silenced cells was determined as compared with controls. These results suggest that MUPP1 specifically, and potentially tight junction complexes in general, are important in the renal osmoadaptive response.


Subject(s)
Carrier Proteins/metabolism , Kidney/metabolism , Tight Junctions/metabolism , Up-Regulation , Adaptation, Biological , Animals , Carrier Proteins/genetics , Cell Line , Cell Survival , Kidney/cytology , Kinetics , Membrane Proteins , Mice , Osmotic Pressure , Proteomics , RNA, Messenger/genetics , Time Factors
5.
J Biol Chem ; 282(9): 6644-52, 2007 Mar 02.
Article in English | MEDLINE | ID: mdl-17200116

ABSTRACT

Proteomic analysis of Inner Medullary Collecting Duct (IMCD3) cells adapted to increasing levels of tonicity (300, 600, and 900 mosmol/kg H(2)O) by two-dimensional difference gel electrophoresis and mass spectrometry revealed several proteins as yet unknown to be up-regulated in response to hypertonic stress. Of these proteins, one of the most robustly up-regulated (22-fold) was S100A4. The identity of the protein was verified by high pressure liquid chromatography-mass spectrometry. Western blot analysis confirmed increased expression with increased tonicity, both acute and chronic. S100A4 protein expression was further confirmed by immunocytochemical analysis. Cells grown in isotonic conditions showed complete absence of immunostaining, whereas chronically adapted IMCD3 cells had uniform cytoplasmic localization. The protein is also regulated in vivo as in mouse kidney tissues S100A4 expression was many -fold greater in the papilla as compared with the cortex and increased further in the papilla upon 36 h of thirsting. Increased expression of S100A4 was also observed in the medulla and papilla, but not the cortex of a human kidney. Data from Affymetrix gene chip analysis and quantitative PCR also revealed increased S100A4 message in IMCD3 cells adapted to hypertonicity. The initial expression of message increased at 8-10 h following exposure to acute sublethal hypertonic stress (550 mosmol/kg H(2)O). Protein and message half-life in IMCD3 cells were 85.5 and 6.8 h, respectively. Increasing medium tonicity with NaCl, sucrose, mannitol, and choline chloride stimulated S100A4 expression, whereas urea did not. Silencing of S100A4 expression using a stable siRNA vector (pSM2; Open Biosystems) resulted in a 48-h delay in adaptation of IMCD3 cells under sublethal osmotic stress, suggesting S100A4 is involved in the osmoadaptive response. In summary, we describe the heretofore unrecognized up-regulation of a small calcium-binding protein, both in vitro and in vivo, whose absence profoundly delays osmoadaptation and slows cellular growth under hypertonic conditions.


Subject(s)
Kidney/physiology , Osmotic Pressure , S100 Proteins/genetics , S100 Proteins/physiology , Up-Regulation/genetics , Adaptation, Physiological , Animals , Calcium-Binding Proteins , Cell Line , Chromatography, High Pressure Liquid , Gene Expression Regulation , Humans , Kidney/cytology , Mass Spectrometry , Mice , Proteomics , S100 Calcium-Binding Protein A4 , S100 Proteins/analysis
6.
J Exp Bot ; 58(5): 1001-11, 2007.
Article in English | MEDLINE | ID: mdl-17220513

ABSTRACT

In mammalian cells, MAPKs are involved in both stress response (JNK and p38 pathways) and cell proliferation and differentiation [extracellular signal-regulated kinase (ERK)] through protein kinase cascades. Exposure of Dunaliella viridis cell cultures to PD98059, a very specific inhibitor of the ERK signalling pathway, resulted in a total arrest of cell proliferation and a complete dephosphorylation of ERK. As shown by flow cytometry analysis of propidium iodide-stained cells, PD98059 stopped mitosis at the G(2) phase after the S phase has been completed. Multiple physiological parameters such as cell motility and reducing power generation (NADPH) clearly indicate that the treated cells are wholly viable. Exposure of D. viridis to environmental stresses that impair cell division, such as hyperosmotic shock, nitrogen starvation, or sublethal UV irradiation, caused a marked decrease in the phospho-ERK levels as detected by western blot. Two 400 bp polynucleotides from D. viridis with high homologies to published sequences of ERK1 and ERK2 were cloned, sequenced, and submitted to GenBank. Northern blot analysis revealed two mRNA bands of approximately 1.9 kb, consistent with the expected size of ERK proteins ( approximately 40 kDa). Sequence analysis showed that they contained several mitogen-activated protein kinase (MAPK) conserved domains, including II, III, VIb, VII, and the double phosphorylation motif. Interestingly, in D. viridis, this motif was T*DY* instead of the canonic T*EY*. Based on this finding, ERK plant sequences can be divided into two groups, one termed the T*DY* branch and the other termed the T*EY* branch. The molecular and functional data presented here suggest that ERK is a very ancient signalling pathway and that it was already present in the last common ancestor of all eukaryotic cells.


Subject(s)
Cell Division/physiology , Eukaryota/cytology , Eukaryota/enzymology , Extracellular Signal-Regulated MAP Kinases/metabolism , Amino Acid Sequence , Cell Division/drug effects , Eukaryota/drug effects , Eukaryota/genetics , Extracellular Signal-Regulated MAP Kinases/chemistry , Extracellular Signal-Regulated MAP Kinases/genetics , Flavonoids/pharmacology , Gene Expression Regulation, Plant/drug effects , Gene Expression Regulation, Plant/physiology , Genes, Plant , Molecular Sequence Data , Phosphorylation , Phylogeny , Plant Proteins/metabolism , RNA, Messenger/metabolism , Time Factors
7.
Am J Physiol Renal Physiol ; 291(6): F1142-7, 2006 Dec.
Article in English | MEDLINE | ID: mdl-16804105

ABSTRACT

The gamma-subunit of Na-K-ATPase is robustly expressed in inner medullary collecting duct (IMCD)3 cells either acutely challenged or adapted to hypertonicity but not under isotonic conditions. Circumstantial evidence suggests that this protein may be important for the survival of renal cells in a hypertonic environment. However, no direct proof for such a contention has been forthcoming. The complete mRNA sequences of either gamma-subunit isoforms were spliced into an expression vector and transfected into IMCD3 cells. Multiple clones stably expressed gamma-subunit protein under isotonic conditions. Clones expressing the gamma(b) isoform showed enhanced survival at lethal acute hypertonicity compared with either gamma(a) isoform or empty vector (control) expressing clones. We also evaluated the loss of gamma-subunit expression on the survival of IMCD3 cells exposed to hypertonicity employing silencing RNA techniques. Multiple stable gamma-subunit-specific siRNA clones were obtained and exposed to sublethal hypertonicity. Under these conditions, both the level of gamma mRNA and protein was essentially undetectable. The impact of silencing gamma-subunit expression resulted in a 70% reduction at 48 h (P < 0.01) in cell survival compared with empty vector (control) clones. gamma siRNA clones showed a 45% decrease in myo-inositol uptake compared with controls after an 18-h exposure to sublethal hypertonicity. Taken together, these data demonstrate a direct and critical role of the gamma-subunit on IMCD3 cell survival and/or adaptation in response to ionic hypertonic stress.


Subject(s)
Gene Silencing , Kidney Tubules, Collecting/cytology , Kidney Tubules, Collecting/physiology , Sodium-Potassium-Exchanging ATPase/genetics , Water-Electrolyte Balance/physiology , Animals , Cell Line , Cell Survival/physiology , Gene Expression , Hypertonic Solutions/pharmacology , Inositol/metabolism , Mice , Osmotic Pressure , RNA, Messenger/metabolism , RNA, Small Interfering , Sodium-Potassium-Exchanging ATPase/metabolism
8.
Front Biosci ; 10: 2604-10, 2005 Sep 01.
Article in English | MEDLINE | ID: mdl-15970522

ABSTRACT

The gamma subunit has been characterized as a fine-tuning modulator of the Na/K-ATPase expressed in kidney tissues. This small single transmembrane domain protein interacts with the alpha subunit of Na/K-ATPase to increase affinity for ATP and decrease affinity for Na allowing medullary cells to continue pump activity under reduced cellular ATP levels. The gamma subunit is undetectable in kidney cell cultures grown under isotonic conditions and expression is induced with acute or chronic exposure to hypertonicity. The gamma subunit demonstrates remarkable regulatory complexity including induction by chloride ions rather than sodium, the differential expression of at least 2 isoforms, involvement of separate MAP kinase signaling pathways for transcription (JNK) and translation (PI3K) as well as cell type regulation of expression. Mutation in the transmembrane domain of the gamma subunit has been implicated in cases of primary hypomagnesemia. Alternative roles have been established for the gamma subunit in embryonic development and quite possibly additional functions in cell signaling as yet unrecognized may be possible.


Subject(s)
Kidney/enzymology , Membrane Proteins/metabolism , Protein Subunits/physiology , Sodium-Potassium-Exchanging ATPase/metabolism , Animals , Cell Line , Gene Expression Regulation , Humans , Membrane Proteins/chemistry , Membrane Proteins/genetics , Protein Subunits/chemistry , Sodium-Potassium-Exchanging ATPase/chemistry , Sodium-Potassium-Exchanging ATPase/genetics
9.
Am J Physiol Renal Physiol ; 288(4): F855-66, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15561974

ABSTRACT

EphA2, a member of the large family of Eph receptor tyrosine kinases, is highly expressed in epithelial tissue and has been implicated in cell-cell and cell-matrix interactions, as well as cell growth and survival. Expression of EphA2 mRNA and protein was markedly upregulated by both hypertonic stress and by elevated urea concentrations in cells derived from the murine inner medullary collecting duct. This upregulation likely required transactivation of the epidermal growth factor (EGF) receptor tyrosine kinase and metalloproteinase-dependent ectodomain cleavage of an EGF receptor ligand, based on pharmacological inhibitor studies. A human EphA2 promoter fragment spanning nucleotides -4030 to +21 relative to the putative EphA2 transcriptional start site was responsive to tonicity but insensitive to urea. A promoter fragment spanning -1890 to +128 recapitulated both tonicity- and urea-dependent upregulation of expression, consistent with transcriptional activation. Neither the bona fide p53 response element at approximately -1.5 kb nor a pair of putative TonE elements at approximately -3 kb conferred the tonicity responsiveness. EphA2 mRNA and protein were expressed at low levels in rat renal cortex but at high levels in the collecting ducts of the renal medulla and papilla. Water deprivation in rats increased EphA2 expression in renal papilla, whereas dietary supplementation with 20% urea increased EphA2 expression in outer medulla. These data indicate that transcription and expression of the EphA2 receptor tyrosine kinase are regulated by tonicity and urea in vitro and suggest that this phenomenon is also operative in vivo. Renal medullary EphA2 expression may represent an adaptive response to medullary hypertonicity or urea exposure.


Subject(s)
Kidney Medulla/physiology , Receptor, EphA2/genetics , Receptor, EphA2/metabolism , Urea/pharmacology , Animals , Cell Line , Gene Expression/drug effects , Humans , Hypertonic Solutions/pharmacology , In Vitro Techniques , Kidney Medulla/cytology , Osmotic Pressure , Phosphorylation , Rats , Rats, Sprague-Dawley , Signal Transduction , Water-Electrolyte Balance/physiology
10.
Am J Physiol Renal Physiol ; 288(1): F76-81, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15383396

ABSTRACT

We previously reported that hypertonicity-mediated upregulation of the gamma-subunit of Na-K-ATPase is dependent on both the JNK and the PI3 kinase pathways. The present experiments were undertaken to explore the mechanisms whereby these pathways regulate the expression of the gamma-subunit in inner medullary collecting duct cells (IMCD3). Inhibition of JNK with SP-600125 (20 muM), a concentration that causes an approximately 95% inhibition of hypertonicity-stimulated JNK activation, markedly decreased the amount of the gamma-subunit in response to 550 mosmol/kgH(2)O for 48 h. This was accompanied by a parallel decrease in the gamma-subunit mRNA. The rate at which the gamma-subunit mRNA decreased was unaffected by actinomycin D. In contrast, inhibition of PI3 kinase with LY-294002 results in a marked decrease in the amount of gamma-subunit protein but without alteration in gamma-subunit message. The rate at which the gamma-subunit protein decreased was unaffected by cyclohexamide. Transfection of IMCD3 cells with a gamma-subunit construct results in the expression of both gamma-subunit message and protein. However, in cortical collecting duct cells (M1 cells) such transfection resulted in expression of only the message and not the protein. We conclude that JNK regulates the gamma-subunit at the transcriptional level while PI3 kinase regulates gamma-subunit expression at the translational level. There is also posttranscriptional cell specificity in the expression of the gamma -subunit of Na-K-ATPase.


Subject(s)
Kidney Medulla/enzymology , Protein Biosynthesis/physiology , Sodium-Potassium-Exchanging ATPase/biosynthesis , Transcription, Genetic/physiology , Animals , Anthracenes/pharmacology , Cell Line , Chromones/pharmacology , Down-Regulation , Gene Expression Regulation, Enzymologic , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , JNK Mitogen-Activated Protein Kinases/metabolism , Kidney Cortex/cytology , Kidney Cortex/enzymology , Kidney Medulla/drug effects , Kidney Tubules, Collecting/drug effects , Kidney Tubules, Collecting/enzymology , MAP Kinase Kinase 4 , Mice , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinase Kinases/metabolism , Morpholines/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Signal Transduction/physiology
11.
Am J Physiol Renal Physiol ; 288(4): F650-7, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15572522

ABSTRACT

Hypertonicity mediated by chloride upregulates the expression of the gamma-subunit of Na-K-ATPase in cultured cells derived from the murine inner medullary collecting duct (IMCD3; Capasso JM, Rivard CJ, Enomoto LM, and Berl T. Proc Natl Acad Sci USA 100: 6428-6433, 2003). The purpose of this study was to examine the cellular locations and the time course of gamma-subunit expression after long-term adaptation and acute hypertonic challenges induced with different salts. Cells were analyzed by confocal immunofluorescence and immunoelectron microscopy with antibodies against the COOH terminus of the Na-K-ATPase gamma-subunit or the gamma(b) splice variant. Cells grown in 300 mosmol/kgH(2)O showed no immunoreactivity for the gamma-subunit, whereas cells adapted to 600 or 900 mosmol/kgH(2)O demonstrated distinct reactivity located at the plasma membrane of all cells. IMCD3 cell cultures acutely challenged to 550 mosmol/kgH(2)O with sodium chloride or choline chloride showed incorporation of gamma into plasma membrane 12 h after osmotic challenge and distinct membrane staining in approximately 40% of the cells 48 h after osmotic shock. In contrast, challenging the IMCD3 cells to 550 mosmol/kgH(2)O by addition of sodium acetate did not result in expression of the gamma-subunit in the membranes of surviving cells after 48 h. The present results demonstrate that the Na-K-ATPase gamma-subunit becomes incorporated into the basolateral membrane of IMCD3 cells after both acute hyperosmotic challenge and hyperosmotic adaptation. We conclude that the gamma-subunit has an important role in the function of Na-K-ATPase to sustain the cellular cation balance over the plasma membrane in a hypertonic environment.


Subject(s)
Cell Membrane/metabolism , Kidney Medulla/enzymology , Sodium-Potassium-Exchanging ATPase/metabolism , Water-Electrolyte Balance/physiology , Adaptation, Physiological , Animals , Blotting, Western , Cell Polarity , Cells, Cultured , Hypertonic Solutions , Immunohistochemistry , Kidney Medulla/cytology , Mice , Osmotic Pressure
12.
Biochim Biophys Acta ; 1644(1): 61-9, 2004 Feb 02.
Article in English | MEDLINE | ID: mdl-14741745

ABSTRACT

The microalga Dunaliella viridis has the ability to adapt to a variety of environmental stresses including osmotic and thermal shocks, UV irradiation and nitrogen starvation. Lacking a rigid cell wall, Dunaliella provides an excellent model to study stress signaling in eukaryotic unicellular organisms. When exposed to hyperosmotic stress, UV irradiation or high temperature, a 57-kDa protein is recognized by antibodies specific to mammalian p38, to its yeast homologue Hog1, and to the phospho-p38 MAP kinase motif. This 57-kDa protein appears to be both up-regulated and phosphorylated. Three other proteins (50, 45, 43 kDa) were transiently phosphorylated under stress conditions as detected with an antibody specific to the mammalian phospho c-Jun N-terminal kinase (JNK) motif. Treatment with specific inhibitors of p38 MAP kinase (SB203580) and JNK (SP600125) activities markedly impaired the adaptation of Dunaliella to osmotic stress. From an evolutionary standpoint, these data strongly suggest that MAP kinase signaling pathways, other than ERK, were already operating in the common ancestor of plant and animal kingdoms, probably as early as 1400 million years ago.


Subject(s)
Chlorophyta/metabolism , JNK Mitogen-Activated Protein Kinases , Mitogen-Activated Protein Kinases/metabolism , Adaptation, Physiological , Antibodies/immunology , Autoradiography , Cells, Cultured , Heat-Shock Proteins/metabolism , Hot Temperature , MAP Kinase Kinase 4 , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinase Kinases/immunology , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/immunology , Osmotic Pressure , Phosphorus Radioisotopes , Phosphorylation , Saline Solution, Hypertonic , Ultraviolet Rays , p38 Mitogen-Activated Protein Kinases
13.
Biochem J ; 378(Pt 3): 919-28, 2004 Mar 15.
Article in English | MEDLINE | ID: mdl-14670077

ABSTRACT

Activation of cellular kinases and transcription factors mediates the early phase of the cellular response to chemically or biologically induced stress. In the present study we investigated the oxidant/antioxidant balance in Huh-7 cells expressing the HCV (hepatitis C virus) subgenomic replicon, and observed a 5-fold increase in oxidative stress during HCV replication. We used MnSOD (manganese-superoxide dismutase) as an indicator of the cellular antioxidant response, and found that its activity, protein levels and promoter activity were significantly increased, whereas Cu/ZnSOD was not affected. The oxidative stress-induced protein kinases p38 MAPK (mitogen-activated protein kinase) and JNK (c-Jun N-terminal kinase) were activated in the HCV repliconcontaining cells and in Huh-7 cells transduced with Ad-NS5A [a recombinant adenovirus encoding NS5A (non-structural protein 5A)], coupled with a 4-5-fold increase in AP-1 (activator protein-1) DNA binding. Ava.1 cells, which encode a replication-defective HCV replicon, showed no significant changes in MnSOD, p38 MAPK or JNK activity. The AP-1 inhibitors dithiothreitol and N -acetylcysteine, as well as a dominant negative AP-1 mutant, significantly reduced AP-1 activation, demonstrating that this activation is oxidative stress-related. Exogenous NS5A had no effect on AP-1 activation in vitro, suggesting that NS5A acts at the upstream targets of AP-1 involving p38 MAPK and JNK signalling cascades. AP-1-dependent gene expression was increased in HCV subgenomic replicon-expressing Huh-7 cells. MnSOD activation was blocked by inhibitors of JNK (JNKI1) and p38 MAPK (SB203580), but not by an ERK (extracellular-signal-regulated kinase) inhibitor (U0126), in HCV-replicating and Ad-NS5A-transduced cells. Our results demonstrate that cellular responses to oxidative stress in HCV subgenomic replicon-expressing and Ad-NS5A-transduced cells are regulated by two distinct signalling pathways involving p38 MAPK and JNK via AP-1 that is linked to increased oxidative stress and therefore to an increased antioxidant MnSOD response.


Subject(s)
Hepacivirus/pathogenicity , Mitogen-Activated Protein Kinases/physiology , Superoxide Dismutase/metabolism , Transcription Factor AP-1/physiology , Cell Line , Enzyme Activation , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Enzymologic , Hepacivirus/genetics , Hepacivirus/physiology , Humans , JNK Mitogen-Activated Protein Kinases , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Oxidative Stress , Promoter Regions, Genetic , Proto-Oncogene Proteins c-jun/biosynthesis , Replicon , Superoxide Dismutase/genetics , Transcriptional Activation , Viral Nonstructural Proteins/metabolism , Virus Replication , p38 Mitogen-Activated Protein Kinases
14.
Biomol Eng ; 20(4-6): 133-8, 2003 Jul.
Article in English | MEDLINE | ID: mdl-12919790

ABSTRACT

In this work, we propose the determination of cell viability in algal cultures by using a colorimetric assay widely used for estimation of cell proliferation in animal cell cultures. The method is based on in vivo reduction by metabolically active cells of a tetrazolium compound (MTS=3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenil)-2H-tetrazolium, inner salt) to a colored formazan, with maximal absorbance at 490 nm, that is released to the culture medium. For this purpose, we have tested two microalgae with high commercial value (Dunaliella and Spirulina) and two seaweeds with different morphology (Ulva and Gracilaria). Color development in this assay is directly proportional to the number of viable cells, to the incubation time in the presence of the assay solution, and to the incubation temperature. A direct significant correlation was found between algal photosynthesis rate and color development in all species used through this work. Moreover, the intensity of absorbance at 490 nm was significantly lower in stressed cells (e.g. in nutrient-limited cultures, in the presence of toxic substances, and in osmotically-stressed cultures). We conclude that cell viability of algal cultures can be rapidly and easily estimated through colorimetric determination of the reduction of MTS to formazan.


Subject(s)
Bioreactors/microbiology , Cell Culture Techniques/methods , Colony Count, Microbial/methods , Colorimetry/methods , Eukaryota/cytology , Eukaryota/metabolism , Formazans/metabolism , Tetrazolium Salts/metabolism , Thiazoles/metabolism , Adaptation, Physiological , Cells, Cultured , Eukaryota/growth & development , Light , Reproducibility of Results , Sensitivity and Specificity , Statistics as Topic , Temperature
15.
Proc Natl Acad Sci U S A ; 100(11): 6428-33, 2003 May 27.
Article in English | MEDLINE | ID: mdl-12746499

ABSTRACT

Hypertonicity induced by NaCl, but not by urea or mannitol, up-regulates expression of the gamma subunit of Na/K-ATPase in cells of the murine inner medullary collecting duct line (IMCD3) by activation of the Jun kinase 2 (JNK2) pathways. We examined the ionic mediators of the osmosensitive response. An increase in osmolality to 550 milliosmoles per kg of water (mosmol/kgH2O) for 48 h by replacement of NaCl with choline chloride did not prevent the up-regulation of the gamma subunit. Neither Na+ ionophores nor inhibitors of cellular Na+ uptake altered the up-regulation of the gamma subunit or JNK activation. Changes in cell cation concentrations driven by incubation in low-K+ medium were effective in up-regulating the alpha1 subunit of Na/K-ATPase but did not have any effect on the gamma subunit. The replacement of NaCl with choline chloride did not down-regulate gamma-subunit expression in cells adapted to hypertonicity. In contrast, the replacement of NaCl with sodium acetate, or pretreatment of cells with the Cl- channel inhibitor 5-nitro-2-(3-phenylpropyl-amino)benzoic acid (NPPB) completely blocked gamma-subunit up-regulation, inhibited JNK activation, and caused a significant decrement in cell survival in hypertonic but not isotonic conditions. In adapted cells, replacement of 300 mosmol/kgH2O NaCl with sodium acetate resulted in down-regulation of the gamma subunit. In conclusion, we describe a Na+-independent, Cl--dependent mechanism for hypertonicity-mediated activation of the JNK and the subsequent synthesis of the gamma subunit of Na/K-ATPase, which are necessary for cellular survival in these anisotonic conditions.


Subject(s)
Chlorides/metabolism , Mitogen-Activated Protein Kinases/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , Animals , Cell Line , Enzyme Activation , Enzyme Inhibitors/pharmacology , JNK Mitogen-Activated Protein Kinases , Mice , Osmolar Concentration , Sodium/metabolism
16.
Ann N Y Acad Sci ; 986: 410-5, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12763858

ABSTRACT

Recently, we have adapted IMCD3 cell cultures to survive under increasing hypertonic conditions (i.e., 600 and 900 mOsmol/kg H(2)O). In adapted cells, ATPase activity is increased by one order of magnitude, while the expression of the alpha and beta subunit is increased by a factor of 4 to 5 over controls (300 mOsmol/kg H(2)O). Corresponding increases in mRNAs were also detected. The gamma subunit has been described as being uniquely expressed in some areas of the kidney, but never in cell cultures (even those derived from kidney tissues). However, the gamma subunit was detected at the protein and mRNA levels in the adapted IMCD3 cells. In contrast to the alpha and beta subunits, the levels of gamma protein and mRNA expression continue to increase as a function of the media ion concentration. We have also demonstrated that signaling pathways that upregulate the alpha, beta, and gamma subunits are very different. Increasing concentrations of the PI3 kinase inhibitor, LY294002, resulted in a dose-dependent reduction in the expression of the gamma subunit, with total abolition at 10 micro M. However, LY294002 had no significant effect on the expression of the alpha subunit. Inhibition of the JNK2 (but not of the JNK1) pathways by dominant negative transfections abolished the upregulation of the gamma, but not the a subunit. Failure to upregulate the expression of the gamma subunit was associated with a marked decrease in cell viability upon stress.


Subject(s)
Kidney Medulla/cytology , Kidney Tubules, Collecting/cytology , Adenosine Triphosphatases/antagonists & inhibitors , Adenosine Triphosphatases/metabolism , Animals , Cell Line , Chromones/pharmacology , Enzyme Inhibitors/pharmacology , Hypertonic Solutions , Kinetics , Mice , Morpholines/pharmacology , Osmolar Concentration , RNA, Messenger/genetics
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