Oxidative stress-induced posttranslational modifications of human hemoglobin in erythrocytes

Posttranslational modifications (PTMs) have been reported in hemoglobin (Hb) treated with ROS/RNS in cell-free experiments. However, little is known about oxidative PTMs of Hb occurring within the erythrocytes. The aim of this study is to characterize the patterns of Hb PTMs in erythrocytes under oxidative stress. Using mass spectrometry, we investigated specifically methionine/tryptophan oxidation, tyrosine nitration, and the modification via 4-hydroxynonenal (HNE), a product of lipid-peroxidation, on Hb. We demonstrated that the treatment with H₂O₂/nitrite induced higher levels of Hb oxidation/nitration in purified Hb preparations than in unpurified hemolysates and erythrocytes, indicating that ROS/RNS are primarily removed by antioxidative mechanisms. We further studied Hb from erythrocytes exposed to γ-irradiation. An irradiation of 30–100 Gy triggered a remarkable increase of intracellular ROS. However, 30 Gy did not induce apparent changes in Hb oxidation/nitration and hemolysis, while Hb oxidation/nitration and hemolysis were significantly enhanced by 100 Gy, suggesting that Hb oxidation/nitration are the consequence of overwhelmed antioxidative mechanisms after oxidative attack and reflect the severity of the oxidative damage of erythrocytes. Although irradiation was known to induce lipid-peroxidation, we could not detect HNE-Hb adducts in irradiated erythrocytes. Analyzing PTM patterns suggests Hb nitration as a more suitable indicator of the oxidative damage of erythrocytes.     

HCl-induced and ATP-dependent upregulation of TRPV1 receptor expression and cytokine production by human esophageal epithelial cells

The pathogenesis of gastroesophageal reflux disease (GERD) remains elusive, but recent evidence suggests that early secretion of inflammatory cytokines and chemokines by the mucosa leads to influx of immune cells followed by tissue damage. We previously showed that exposure of esophageal mucosa to HCl causes ATP release, resulting in activation of acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine acetyltransferase (lyso-PAF AT), the enzyme responsible for the production of platelet-activating factor (PAF). In addition, HCl causes release of IL-8 from the esophageal mucosa. We demonstrate that esophageal epithelial cells secrete proinflammatory mediators in response to HCl and that this response is mediated by ATP. Monolayers of the human esophageal epithelial cell line HET-1A were exposed to acidified cell culture medium (pH 5) for 12 min, a total of seven times over 48 h, to simulate the recurrent acid exposure clinically occurring in GERD. HCl upregulated mRNA and protein expression for the acid-sensing transient receptor potential cation channel, subfamily vanilloid member 1 (TRPV1), lyso-PAF AT, IL-8, eotaxin-1, -2, and -3, macrophage inflammatory protein-1α, and monocyte chemoattractant protein-1. The chemokine profile secreted by HET-1A cells in response to repeated HCl exposure parallels similar findings in erosive esophagitis patients. In HET-1A cells, the TRPV1 agonist capsaicin reproduced these findings for mRNA of the inflammatory mediators lyso-PAF AT, IL-8, and eotaxin-1. These effects were blocked by the TRPV1 antagonists iodoresiniferatoxin and JNJ-17203212. These effects were imitated by direct application of ATP and blocked by the nonselective ATP antagonist suramin. We conclude that HCl/TRPV-induced ATP release upregulated secretion of various chemoattractants by esophageal epithelial cells. These chemoattractants are selective for leukocyte subsets involved in acute inflammatory responses and allergic inflammation. The data support the validity of HET-1A cells as a model of the response of the human esophageal mucosa in GERD.     

TRPV3 endogenously expressed in murine colonic epithelial cells is inhibited by the novel TRPV3 blocker 26E01

TRPV3 is a Ca²⁺-permeable cation channel, prominently expressed by keratinocytes where it contributes to maintaining the skin barrier, skin regeneration, and keratinocyte differentiation. However, much less is known about its physiological function in other tissues and there is still a need for identifying novel and efficient TRPV3 channel blockers. By screening a compound library, we identified 26E01 as a novel TRPV3 blocker. 26E01 blocks heterologously expressed TRPV3 channels overexpressed in HEK293 cells as assessed by fluorometric intracellular free Ca²⁺ assays (IC₅₀ = 8.6 μM) but does not affect TRPV1, TRPV2 or TRPV4 channels. Electrophysiological whole-cell recordings confirmed the reversible block of TRPV3 currents by 26E01, which was also effective in excised inside-out patches, hinting to a rather direct mode of action. 26E01 suppresses endogenous TRPV3 currents in the mouse 308 keratinocyte cell line and in the human DLD-1 colon carcinoma cell line (IC₅₀ = 12 μM). In sections of the gastrointestinal epithelium of mice, the expression of TRPV3 mRNA follows a gradient along the gastrointestinal tract, with the highest expression in the distal colon. 26E01 efficiently attenuates 2-aminoethoxydiphenyl borate-induced calcium influx in primary colonic epithelial cells isolated from the distal colon. As 26E01 neither shows toxic effects on DLD-1 cells at concentrations of up to 100 μM in MTT assays nor on mouse primary colonic crypts as assessed by calcein-AM/propidium iodide co-staining, it may serve as a useful tool to further study the physiological function of TRPV3 in various tissues.     

Oxidative stress-induced apoptosis of endothelial cells

Endothelial cells (ECs) are subjected to oxidative stress during many pathological processes, including ischemia/reperfusion and general inflammation. In the present study, we examined the effects of oxidative stress on rates of apoptosis in EC cultures. We treated large and microvessel ECs with menadione for 1 h in vitro to simulate the most common physiological form of oxidative stress, exposure to O2*-. Capillary ECs were resistant to menadione-induced apoptosis when compared with large-vessel ECs. Treatment with 35 microM menadione resulted in an apoptotic rate of approximately 5% in capillary EC cultures compared with approximately 45% in large-vessel EC cultures. At higher concentrations of menadione (35-75 microM), both types of ECs exhibited a concentration-related increase in apoptosis. Necrotic cell death only became evident at menadione concentrations ranging from 75-100 microM for both cell types. The timing of the apoptotic response to a 1 h menadione exposure was very specific. For both EC types, peaks of apoptosis occurred in two distinct waves, at 6-8 and 18-22 h after treatment. Analysis of the events leading up to the first peak of apoptosis indicated that specific matrix metalloproteinases (MMPs) were activated, suggesting that MMPs may be involved in initiating the apoptotic process.     

The posttranslational modification of tubulin undergoes a switch from detyrosination to acetylation as epithelial cells become polarized

Tubulin posttranslational modifications (PTMs) have been suggested to provide navigational cues for molecular motors to deliver cargo to spatially segregated subcellular domains, but the molecular details of this process remain unclear. Here we show that in Madin-Darby Canine Kidney (MDCK) epithelial cells, microtubules express several tubulin PTMs. These modifications, however, are not coordinated, and cells have multiple subpopulations of microtubules that are marked by different combinations of PTMs. Furthermore these subpopulations show differential sensitivity to both drug- and cold-induced depolymerization, suggesting that they are functionally different as well. The composition and distribution of modified microtubules change as cells undergo the morphogenesis associated with polarization. Two-dimensionally polarized spreading cells have more detyrosinated microtubules that are oriented toward the leading edge, but three-dimensionally polarized cells have more acetylated microtubules that are oriented toward the apical domain. These data suggest that the transition from 2D polarity to 3D polarity involves both a reorganization of the microtubule cytoskeleton and a change in tubulin PTMs. However, in both 2D polarized and 3D polarized cells, the modified microtubules are oriented to support vectorial cargo transport to areas of high need.     

The TRPV4 channel is a novel regulator of intracellular Ca2+ in human esophageal epithelial cells

The esophageal epithelium has sensory properties that enable it to sustain normal barrier function. Transient receptor potential vanilloid 4 (TRPV4) is a Ca(2+)-permeable channel that is activated by extracellular hypotonicity, polyunsaturated fatty acids, phorbol esters, and elevated temperature. We found that TRPV4 is expressed in both human esophageal tissue and in HET-1A cells, a human esophageal epithelial cell line. Specific activation of TRPV4 by the phorbol ester 4α-phorbol 12,13-didecanoate (4α-PDD) increased intracellular Ca(2+) in a subset of HET-1A cells. Elevated temperature strongly potentiated this effect at low concentrations of 4α-PDD, and all of the responses were inhibited by the TRPV antagonist ruthenium red. TRPV4 activation differentially affected cell proliferation and cell viability; HET-1A cell proliferation was increased by 1 μM 4α-PDD, whereas higher concentrations (10 μM and 30 μM) significantly decreased cell viability. Transient TRPV4 activation triggered ATP release in a concentration-dependent manner via gap-junction hemichannels, including pannexin 1 and connexin 43. Furthermore, TRPV4 activation for 24 h did not increase the production of interleukin 8 (IL-8) but reduced IL-1β-induced IL-8 production. Small-interference RNA targeted to TRPV4 significantly attenuated all of the 4α-PDD-induced responses in HET-1A cells. Collectively, these findings suggest that TRPV4 is a novel regulator of Ca(2+)-dependent signaling pathways linked to cell proliferation, cell survival, ATP release, and IL-8 production in human esophageal epithelial cells.     

Romo1 expression contributes to oxidative stress-induced death of lung epithelial cells

Oxidant-mediated death of lung epithelial cells due to cigarette smoking plays an important role in pathogenesis in lung diseases such as idiopathic pulmonary fibrosis (IPF). However, the exact mechanism by which oxidants induce epithelial cell death is not fully understood. Reactive oxygen species (ROS) modulator 1 (Romo1) is localized in the mitochondria and mediates mitochondrial ROS production through complex III of the mitochondrial electron transport chain. Here, we show that Romo1 mediates mitochondrial ROS production and apoptosis induced by oxidative stress in lung epithelial cells. Hydrogen peroxide (H₂O₂) treatment increased Romo1 expression, and Romo1 knockdown suppressed the cellular ROS levels and cell death triggered by H₂O₂ treatment. In immunohistochemical staining of lung tissues from patients with IPF, Romo1 was mainly localized in hyperplastic alveolar and bronchial epithelial cells. Romo1 overexpression was detected in 14 of 18 patients with IPF. TUNEL-positive alveolar epithelial cells were also detected in most patients with IPF but not in normal controls. These findings suggest that Romo1 mediates apoptosis induced by oxidative stress in lung epithelial cells.     

Galectin-1 is expressed by thymic epithelial cells in myasthenia gravis

Galectin-1, a member of a family of carbohydrate binding proteins, is synthesized by thymic epithelial cells in normal juvenile thymus, and mediates adhesion of immature T cells to thymic epithelium. Because cell adhesion molecules are proposed to play a role in the thymic hyperplasia and neoplasia seen in the autoimmune disease myasthenia gravis, we examined the expression of galectin-1 in myasthenic thymi. We detected abundant galectin-1 expression in thymic epithelial cells in 27 hyperplastic and neoplastic thymi from patients with myasthenia gravis. Primary cultures of neoplastic epithelial cells from a thymoma continued to express galectin-1, and bound immature T cells; T cell binding was inhibited by the addition of the -galactosides lactose and thiodigalactoside, suggesting that galectin-1 on the thymoma cells and a saccharide ligand on the T cells participated in cell-cell adhesion. Expression of galectin-1 by thymic epithelial cells may play a role in the thymic pathology seen in myasthenia gravis.     

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.     

A major posttranslational modification of ezrin takes place during epithelial differentiation in the early mouse embryo

The preimplantation development of the mouse embryo leads to the formation of two populations of cells: the trophectoderm, which is a perfect epithelium, and the inner cell mass. The divergence between these two lineages is the result of asymmetric divisions, which can occur after blastomere polarization at compaction. The apical pole of microvilli is the only asymmetric feature maintained during mitosis and polarity is reestablished only in daughter cells that inherit all or a sufficient part of this pole. To analyze the role of ezrin in the formation and stabilization of the pole of microvilli, we isolated and cultured inner cell masses (ICM). These undifferentiated cells can differentiate very quickly into epithelial cells. After isolation of the ICMs, ezrin relocalizes at the cell cortex before the formation of microvilli. This redistribution occurs in the absence of protein synthesis. The formation of microvilli at the apical surface of the outer cells of ICM correlates with a major posttranslational modification of ezrin. We show here that this posttranslational modification is not controlled by a serine/threonine kinase but an O-glycosylation may partially contribute to it. These data suggest that ezrin has at least two roles during development. First, ezrin may be involved in the formation of microvilli because it localizes at the cell cortex before microvilli appear in ICMs. Second, ezrin may stabilize the pole of microvilli because it is modified posttranslationally when microvilli form.     

A novel transglutaminase-catalyzed posttranslational modification of HIV-1 aspartyl protease.

We demonstrate that HIV-1 aspartyl protease (AP), the enzyme essential for the maturation of the AIDS virus, covalently incorporates spermidine catalyzed by guinea pig liver transglutaminase (TGase) and human coagulation factor XIIIa. Preliminary evidence indicates that there are at least three reactive glutamyl and lysyl residues in AP which act as acyl donor and acceptor respectively in a TGase reaction. SDS-PAGE and chromatographic analyses indicate that the two TGases tested catalyze the incorporation of radioactive spermidine into pure HIV-1 AP. The chemical identification and quantitation of (gamma-glutamyl) spermidine isopeptide provide conclusive evidence that the formation of this derivative is catalyzed by TGase. These results imply that TGase-catalyzed post-translational modification of HIV-1 AP may take place in a manner similar to the ones demonstrated in porcine pancreatic phospholipase A2.     

Chloride transport in rabbit esophageal epithelial cells

We investigated Cl(-) transport pathways in the apical and basolateral membranes of rabbit esophageal epithelial cells (EEC) using conventional and ion-selective microelectrodes. Intact sections of esophageal epithelium were mounted serosal or luminal side up in a modified Ussing chamber, where transepithelial potential difference and transepithelial resistance could be determined. Microelectrodes were used to measure intracellular Cl(-) activity (a), basolateral or apical membrane potentials (V(mBL) or V(mC)), and the voltage divider ratio. When a basal cell was impaled, V(mBL) was -73 +/- 4.3 mV and a(i)(Cl) was 16.4 +/- 2.1 mM, which were similar in presence or absence of bicarbonate. Removal of serosal Cl(-) caused a transient depolarization of V(mBL) and a decrease in a(i)(Cl) of 6.5 +/- 0.9 mM. The depolarization and the rate of decrease of a(i)(Cl) were inhibited by approximately 60% in the presence of the Cl(-)-channel blocker flufenamate. Serosal bumetanide significantly decreased the rate of change of a(i)(Cl) on removal and readdition of serosal Cl(-). When a luminal cell was impaled, V(mC) was -65 +/- 3.6 mV and a was 16.3 +/- 2.2 mM. Removal of luminal Cl(-) depolarized V(mC) and decreased a by only 2.5 +/- 0.9 mM. Subsequent removal of Cl(-) from the serosal bath decreased a(i)(Cl) in the luminal cell by an additional 6.4 +/- 1.0 mM. A plot of V(mBL) measurements vs. log a(i)(Cl)/log a(o)(Cl) (a(o)(Cl) is the activity of Cl(-) in a luminal or serosal bath) yielded a straight line [slope (S) = 67.8 mV/decade of change in a(i)(Cl)/a(o)(Cl)]. In contrast, V(mC) correlated very poorly with log a/a (S = 18.9 mV/decade of change in a/a). These results indicate that 1) in rabbit EEC, a(i)(Cl) is higher than equilibrium across apical and basolateral membranes, and this process is independent of bicarbonate; 2) the basolateral cell membrane possesses a conductive Cl(-) pathway sensitive to flufenamate; and 3) the apical membrane has limited permeability to Cl(-), which is consistent with the limited capacity for transepithelial Cl(-) transport. Transport of Cl(-) at the basolateral membrane is likely the dominant pathway for regulation of intracellular Cl(-).     

Characterization of a novel posttranslational modification in polypyrimidine tract-binding proteins by SUMO1

Polypyrimidine tract-binding protein 1 (PTBP1) and its brainspecific homologue, PTBP2, are associated with pre-mRNAs and influence pre-mRNA processing, as well as mRNA metabolism and transport. They play important roles in neural differentiation and glioma development. In our study, we detected the expression of the two proteins in glioma cells and predicted that they may be sumoylated using SUMOplot analyses. We confirmed that PTBP1 and PTBP2 can be modified by SUMO1 with co-immunoprecipitation experiments using 293ET cells transiently co-expressing SUMO1 and either PTBP1 or PTBP2. We also found that SUMO1 modification of PTBP2 was enhanced by Ubc9 (E2). The mutation of the sumoylation site (Lys137) of PTBP2 markedly inhibited its modification by SUMO1. Interestingly, in T98G glioma cells, the level of sumoylated PTBP2 was reduced compared to that of normal brain cells. Overall, this study shows that PTBP2 is posttranslationally modified by SUMO1. [BMB Reports 2014; 47(4): 233-238]     

Oxidative stress-induced phospholipase C-gamma 1 activation enhances cell survival

Phospholipase C-gamma1 (PLC-gamma1) is rapidly activated in response to growth factor stimulation and plays an important role in regulating cell proliferation and differentiation through the generation of the second messengers diacylglycerol and inositol 1,4,5-trisphosphate, leading to the activation of protein kinase C (PKC) and increased levels of intracellular calcium, respectively. Given the existing overlap between signaling pathways that are activated in response to oxidant injury and those involved in responding to proliferative stimuli, we investigated the role of PLC-gamma1 during the cellular response to oxidative stress. Treatment of normal mouse embryonic fibroblasts (MEF) with H2O2 resulted in time- and concentration-dependent tyrosine phosphorylation of PLC-gamma1. Phosphorylation could be blocked by pharmacological inhibitors of Src family tyrosine kinases or the epidermal growth factor receptor tyrosine kinase, but not by inhibitors of the platelet-derived growth factor receptor or phosphatidylinositol 3-kinase. To investigate the physiologic relevance of H2O2-induced tyrosine phosphorylation of PLC-gamma1, we compared survival of normal MEF and PLC-gamma1-deficient MEF following exposure to H2O2. Treatment of PLC-gamma1-deficient MEF with H2O2 resulted in rapid cell death, whereas normal MEF were resistant to the stress. Pretreatment of normal MEF with a selective pharmacological inhibitor of PLC-gamma1, or inhibitors of inositol trisphosphate receptors and PKC, increased their sensitivity to H2O2, whereas treatment of PLC-gamma1-deficient MEF with agents capable of directly activating PKC and enhancing calcium mobilization significantly improved their survival. Finally, reconstitution of PLC-gamma1 protein expression in PLC-gamma1-deficient MEF restored cell survival following H2O2 treatment. These findings suggest an important protective function for PLC-gamma1 activation during the cellular response to oxidative stress.     

Functional TRPV4 channels are expressed in human airway smooth muscle cells

Hypotonic stimulation induces airway constriction in normal and asthmatic airways. However, the osmolarity sensor in the airway has not been characterized. TRPV4 (also known as VR-OAC, VRL-2, TRP12, OTRPC4), an osmotic-sensitive cation channel in the transient receptor potential (TRP) channel family, was recently cloned. In the present study, we show that TRPV4 mRNA was expressed in cultured human airway smooth muscle cells as analyzed by RT-PCR. Hypotonic stimulation induced Ca(2+) influx in human airway smooth muscle cells in an osmolarity-dependent manner, consistent with the reported biological activity of TRPV4 in transfected cells. In cultured muscle cells, 4alpha-phorbol 12,13-didecanoate (4-alphaPDD), a TRPV4 ligand, increased intracellular Ca(2+) level only when Ca(2+) was present in the extracellular solution. The 4-alphaPDD-induced Ca(2+) response was inhibited by ruthenium red (1 microM), a known TRPV4 inhibitor, but not by capsazepine (1 microM), a TRPV1 antagonist, indicating that 4-alphaPDD-induced Ca(2+) response is mediated by TRPV4. Verapamil (10 microM), an L-type voltage-gated Ca(2+) channel inhibitor, had no effect on the 4-alphaPDD-induced Ca(2+) response, excluding the involvement of L-type Ca(2+) channels. Furthermore, hypotonic stimulation elicited smooth muscle contraction through a mechanism dependent on membrane Ca(2+) channels in both isolated human and guinea pig airways. Hypotonicity-induced airway contraction was not inhibited by the L-type Ca(2+) channel inhibitor nifedipine (1 microM) or by the TRPV1 inhibitor capsazepine (1 microM). We conclude that functional TRPV4 is expressed in human airway smooth muscle cells and may act as an osmolarity sensor in the airway.     

Towards posttranslational modification proteome of royal jelly

Royal jelly (RJ) is a secretory protein from the hypopharyngeal glands of nurse honeybee workers, which contains a variety of proteins of which major royal jelly proteins (MRJPs) are some of the most important. It plays important roles both for honeybee and human. Each family of MRJP 1-5 displays a string of modified protein spots in the RJ proteome profile, which may be caused by posttranslational modifications (PTMs) of MRJPs. However, information on the RJ PTMs is still limited. Therefore, the PTM status of RJ was identified by using complementary proteome strategies of two-dimensional gel electrophoresis (2-DE), shotgun analysis in combination with high performance liquid chromatography-chip/electrospray ionization quadrupole time-of-flight/tandem mass spectrometry and bioinformatics. Phosphorylation was characterized in MRJP 1, MRJP 2 and apolipophorin-III-like protein for the first time and a new site was localized in venom protein 2 precursor. Methylation and deamidation were also identified in most of the MRJPs. The results indicate that methylation is the most important PTM of MRJPs that triggers the polymorphism of MRJP 1-5 in the RJ proteome. Our data provide a comprehensive catalog of several important PTMs in RJ and add valuable information towards assessing both the biological roles of these PTMs and deciphering the mechanisms underlying the beneficial effects of RJ for human health.     

Slow posttranslational modification of a neurofilament protein

The synthesis and subsequent modification of neurofilament (NF) polypeptides has been examined in pulse-chase experiments, using cultured chick spinal cord neurons. Fluorography of the [35S]methionine-labeled cytoskeletal proteins, after separation by two-dimensional gel electrophoresis, revealed that (a) the mid-size chicken NF protein, NF-M160, is synthesized as a smaller and more basic precursor, NF-M130; (b) beginning approximately 8 h after translation, NF-M130 slowly and continuously becomes larger and more acidic, attaining the size and charge of NF-M160 16 or more h later, and undergoing no further change in mobility for many days thereafter; and (c) in contrast, the low molecular weight NF protein, NF-L, is synthesized as such, and undergoes no subsequent change in apparent size or charge. Additional experiments provided evidence that the conversion of NF-M130 to NF-M160 is due, at least in part, to phosphorylation: (a) Incubation of similar cultures in 32PO4 resulted in incorporation into NF-M160 and transitional forms, but not into NF-M130. (b) An antiserum to NF-M160 was found by immunoblot analysis to bind strongly to untreated NF-M160, but poorly to phosphatase-treated NF-M160, and not at all to NF-M130. It has already been demonstrated (Bennett, G. S., S. J. Tapscott, C. DiLullo, and H. Holtzer, 1984, Brain Res., 304:291-302) that this anti-NF-M160 fails to stain the soma of motor neurons in sections of chick spinal cord, but detects an increasing gradient of immunoreactivity in the proximal axons. These results, together with the known kinetics of axoplasmic transport of NF, suggest that the mid-size chicken NF protein is synthesized as NF-M130 and is extensively modified, at least in part by phosphorylation, to become NF-M160 during transport along proximal neurites. Once maximally modified, NF-M160 undergoes no further net change during transport along distal neurites.     

Oxidative stress-induced calcium signaling in Arabidopsis

Many environmental stresses result in increased generation of active oxygen species in plant cells. This leads to the induction of protective mechanisms, including changes in gene expression, which lead to antioxidant activity, the recovery of redox balance, and recovery from damage/toxicity. Relatively little is known about the signaling events that link perception of increased active oxygen species levels to gene expression in plants. We have investigated the role of calcium signaling in H2O2-induced expression of the GLUTATHIONE-S-TRANSFERASE1 (GST1) gene. Challenge with H2O2 triggered a biphasic Ca2+ elevation in Arabidopsis seedlings. The early Ca2+ peak localized to the cotyledons, whereas the late Ca2+ rise was restricted to the root. The two phases of the Ca2+ response were independent of each other, as shown by severing shoot from root tissues before H2O2 challenge. Modulation of the height of Ca2+ rises had a corresponding effect upon H2O2-induced GST1 expression. Application of the calcium channel blocker lanthanum reduced the height of the first Ca2+ peak and concomitantly inhibited GST1 expression. Conversely, enhancing the height of the H2O2-triggered Ca2+ signature by treatment with L-buthionine-[S,R]-sulfoximine (an inhibitor of glutathione synthesis) lead to enhancement of GST1 induction. This finding also indicates that changes in the cellular redox balance constitute an early event in H2O2 signal transduction as reduction of the cellular redox buffer and thus the cell's ability to maintain a high GSH/GSSG ratio potentiated the plant's antioxidant response.