Stress-induced Gene Expression Research Articles

Sumoylation of the THO complex regulates the biogenesis of a subset of mRNPs

Assembly of messenger ribonucleoparticles (mRNPs) is a pivotal step in gene expression, but only a few molecular mechanisms contributing to its regulation have been described. Here, through a comprehensive proteomic survey of mRNP assembly, we demonstrate that the SUMO pathway specifically controls the association of the THO complex with mRNPs. We further show that the THO complex, a key player in the interplay between gene expression, mRNA export and genetic stability, is sumoylated on its Hpr1 subunit and that this modification regulates its association with mRNPs. Altered recruitment of the THO complex onto mRNPs in sumoylation-defective mutants does not affect bulk mRNA export or genetic stability, but impairs the expression of acidic stress-induced genes and, consistently, compromises viability in acidic stress conditions. Importantly, inactivation of the nuclear exosome suppresses the phenotypes of the hpr1 non-sumoylatable mutant, showing that SUMO-dependent mRNP assembly is critical to allow a specific subset of mRNPs to escape degradation. This article thus provides the first example of a SUMO-dependent mRNP-assembly event allowing a refined tuning of gene expression, in particular under specific stress conditions.

Promoters of HcTPS1 and HcTPS2 Genes from Hedychium coronarium Direct Floral-Specific, Developmental-Regulated and Stress-Inducible Gene Expression in Transgenic Tobacco

HcTPS1 and HcTPS2 are floral-scent genes that mediate terpene formation in Hedychium coronarium. Transcription of these genes is induced by various abiotic stresses and hormonal stimuli. In this study, the regulatory mechanisms responsible for HcTPS1 and HcTPS2 expression were investigated by analyzing their promoters. Two genomic fragments, 1,298-bp and 1,928-bp in length, upstream of HcTPS1 and HcTPS2, respectively, were isolated and identified as the promoters ‘PrHcTPS1’ and ‘PrHcTPS2’. Analysis of both promoter sequences indicated cis-elements associated with abiotic stress and jasmonic acid (JA) responses, as well as tissue-specific expression. To identify the functional regions within each of the two promoters, a series of 5′ deletion fragments of different lengths from each promoter were fused to a β-glucuronidase (GUS) reporter gene (pCAMBIA1300-‘prHcTPS’:: gusA) and subsequently used to transform Nicotiana tobacum L. Wisconsin (w38). Functional properties of each promoter segment were determined by GUS staining and quantitative fluorescence analyses of transgenic plants subjected to various abiotic stresses and plant hormones. GUS histochemical staining in transgenic tobacco containing the full fragment of either promoter sequence was only observed in floral organs, where gusA transcripts accumulated as flower development progressed. Additionally, gusA transcripts were also up-regulated by insect feeding, methyl jasmonate (MeJA), and wounding treatments, indicating that ‘PrHcTPS1’ and ‘PrHcTPS2’ promoters were floral-specific, developmentally-regulated, and stress-inducible. Promoter deletion analysis indicated that the promoter fragments p-420 bp of ‘PrHcTPS1’ and p-320 bp of ‘PrHcTPS2’ possessed sufficient, essential cis-elements to drive the expression patterns observed using the whole fragment of either promoter. The identified promoters could be extremely valuable for future use in molecular breeding of ornamental and horticultural plants for floral scent and stress tolerance.

Promoter‐associated histone acetylation is involved in the osmotic stress‐induced transcriptional regulation of the maize ZmDREB2A gene

Epigenetic modifications play a key role in the transcriptional regulation of stress-induced gene expression in plants. In this study, we showed that the overall acetylation levels of histone H3 lysine 9 (H3K9) and H4 lysine 5 (H4K5) in the genome were increased in maize seedlings after mannitol treatment (to mimic osmotic stress). Mannitol treatment significantly induced the upregulation of the maize osmotic stress responsive gene Zea mays dehydration-responsive element binding protein 2A (ZmDREB2A), whereas abscisic acid (ABA) did not result in the induction of this gene. The application of exogenous ABA under osmotic stress conditions strongly repressed the induction of the ZmDREB2A gene. Chromatin immunoprecipitation and chromatin accessibility by real-time PCR experiments revealed that the promoter region of the ZmDREB2A gene was quickly hyperacetylated and decondensed after the mannitol treatment, suggesting that the promoter region is poised for histone acetylation to allow for fast induction of the ZmDREB2A gene. However, under osmotic stress conditions, the ABA treatment decreased the acetylation status and chromatin accessibility to micrococcal nuclease. These results suggest that osmotic stress activates the transcription of the ZmDREB2A gene by increasing the levels of acetylated histones H3K9 and H4K5 associated with the ZmDREB2A promoter region.

25-Hydroxycholesterol Activates the Integrated Stress Response to Reprogram Transcription and Translation in Macrophages

25-Hydroxycholesterol (25OHC) is an enzymatically derived oxidation product of cholesterol that modulates lipid metabolism and immunity. 25OHC is synthesized in response to interferons and exerts broad antiviral activity by as yet poorly characterized mechanisms. To gain further insights into the basis for antiviral activity, we evaluated time-dependent responses of the macrophage lipidome and transcriptome to 25OHC treatment. In addition to altering specific aspects of cholesterol and sphingolipid metabolism, we found that 25OHC activates integrated stress response (ISR) genes and reprograms protein translation. Effects of 25OHC on ISR gene expression were independent of liver X receptors and sterol-response element-binding proteins and instead primarily resulted from activation of the GCN2/eIF2α/ATF4 branch of the ISR pathway. These studies reveal that 25OHC activates the integrated stress response, which may contribute to its antiviral activity.

PERK Activation Preserves the Viability and Function of Remyelinating Oligodendrocytes in Immune-Mediated Demyelinating Diseases

Remyelination occurs in multiple sclerosis (MS) lesions but is generally considered to be insufficient. One of the major challenges in MS research is to understand the causes of remyelination failure and to identify therapeutic targets that promote remyelination. Activation of pancreatic endoplasmic reticulum kinase (PERK) signaling in response to endoplasmic reticulum stress modulates cell viability and function under stressful conditions. There is evidence that PERK is activated in remyelinating oligodendrocytes in demyelinated lesions in both MS and its animal model, experimental autoimmune encephalomyelitis (EAE). In this study, we sought to determine the role of PERK signaling in remyelinating oligodendrocytes in MS and EAE using transgenic mice that allow temporally controlled activation of PERK signaling specifically in oligodendrocytes. We demonstrated that persistent PERK activation was not deleterious to myelinating oligodendrocytes in young, developing mice or to remyelinating oligodendrocytes in cuprizone-induced demyelinated lesions. We found that enhancing PERK activation, specifically in (re)myelinating oligodendrocytes, protected the cells and myelin against the detrimental effects of interferon-γ, a key proinflammatory cytokine in MS and EAE. More important, we showed that enhancing PERK activation in remyelinating oligodendrocytes at the recovery stage of EAE promoted cell survival and remyelination in EAE demyelinated lesions. Thus, our data provide direct evidence that PERK activation cell-autonomously enhances the survival and preserves function of remyelinating oligodendrocytes in immune-mediated demyelinating diseases.

Are Interstitial Cells of Cajal Involved in Mechanical Stress-Induced Gene Expression and Impairment of Smooth Muscle Contractility in Bowel Obstruction?

Background and AimsThe network of interstitial cells of Cajal (ICC) is altered in obstructive bowel disorders (OBD). However, whether alteration in ICC network is a cause or consequence of OBD remains unknown. This study tested the hypothesis that mechanical dilation in obstruction disrupts the ICC network and that ICC do not mediate mechanotranscription of COX-2 and impairment of smooth muscle contractility in obstruction.MethodsMedical-grade silicon bands were wrapped around the distal colon to induce partial obstruction in wild-type and ICC deficient (W/Wv) mice.ResultsIn wild-type mice, colon obstruction led to time-dependent alterations of the ICC network in the proximal colon segment. Although unaffected on days 1 and 3, the ICC density decreased markedly and the network was disrupted on day 7 of obstruction. COX-2 expression increased, and circular muscle contractility decreased significantly in the segment proximal to obstruction. In W/Wv control mice, COX-2 mRNA level was 4.0 (±1.1)-fold higher (n=4) and circular muscle contractility was lower than in wild-type control mice. Obstruction further increased COX-2 mRNA level in W/Wv mice to 7.2 (±1.0)-fold vs. W/Wv controls [28.8 (±4.1)-fold vs. wild-type controls] on day 3. Obstruction further suppressed smooth muscle contractility in W/Wv mice. However, daily administration of COX-2 inhibitor NS-398 significantly improved muscle contractility in both W/Wv sham and obstruction mice.ConclusionsLumen dilation disrupts the ICC network. ICC deficiency has limited effect on stretch-induced expression of COX-2 and suppression of smooth muscle contractility in obstruction. Rather, stretch-induced COX-2 plays a critical role in motility dysfunction in partial colon obstruction.

Inhibition of Inducible Heat Shock Protein-70 (Hsp72) Enhances Bortezomib-Induced Cell Death in Human Bladder Cancer Cells

The proteasome inhibitor bortezomib (Velcade) is a promising new agent for bladder cancer therapy, but inducible cytoprotective mechanisms may limit its potential efficacy. We used whole genome mRNA expression profiling to study the effects of bortezomib on stress-induced gene expression in a panel of human bladder cancer cell lines. Bortezomib induced strong upregulation of the inducible HSP70 isoforms HSPA1A and HSPA1B isoforms of Hsp72 in 253J B-V and SW780 (HSPA1Ahigh) cells, but only induced the HSPA1B isoform in UM-UC10 and UM-UC13 (HSPA1Alow) cells. Bortezomib stimulated the binding of heat shock factor-1 (HSF1) to the HSPA1A promoter in 253JB-V but not in UM-UC13 cells. Methylation-specific PCR revealed that the HSPA1A promoter was methylated in the HSPA1Alow cell lines (UM-UC10 and UM-UC13), and exposure to the chromatin demethylating agent 5-aza-2′-deoxycytidine restored HSPA1A expression. Overexpression of Hsp72 promoted bortezomib resistance in the UM-UC10 and UM-UC13 cells, whereas transient knockdown of HSPA1B further sensitized these cells to bortezomib, and exposure to the chemical HSF1 inhibitor KNK-437 promoted bortezomib sensitivity in the 253J B-V cells. Finally, shRNA-mediated stable knockdown of Hsp72 in 253J B–V promoted sensitivity to bortezomib in vitro and in tumor xenografts in vivo. Together, our results provide proof-of-concept for using Hsp72 inhibitors to promote bortezomib sensitivity in bladder cancers and suggest that selective targeting of HSPA1B could produce synthetic lethality in tumors that display HSPA1A promoter methylation.

The Arabidopsis RNA Binding Protein with K Homology Motifs, SHINY1, Interacts with the C-terminal Domain Phosphatase-like 1 (CPL1) to Repress Stress-Inducible Gene Expression

The phosphorylation state of the C-terminal domain (CTD) of the RNA polymerase II plays crucial roles in transcription and mRNA processing. Previous studies showed that the plant CTD phosphatase-like 1 (CPL1) dephosphorylates Ser-5-specific CTD and regulates abiotic stress response in Arabidopsis. Here, we report the identification of a K-homology domain-containing protein named SHINY1 (SHI1) that interacts with CPL1 to modulate gene expression. The shi1 mutant was isolated from a forward genetic screening for mutants showing elevated expression of the luciferase reporter gene driven by a salt-inducible promoter. The shi1 mutant is more sensitive to cold treatment during vegetative growth and insensitive to abscisic acid in seed germination, resembling the phenotypes of shi4 that is allelic to the cpl1 mutant. Both SHI1 and SHI4/CPL1 are nuclear-localized proteins. SHI1 interacts with SHI4/CPL1 in vitro and in vivo. Loss-of-function mutations in shi1 and shi4 resulted in similar changes in the expression of some stress-inducible genes. Moreover, both shi1 and shi4 mutants display higher mRNA capping efficiency and altered polyadenylation site selection for some of the stress-inducible genes, when compared with wild type. We propose that the SHI1-SHI4/CPL1 complex inhibits transcription by preventing mRNA capping and transition from transcription initiation to elongation.

The eIF2α/ATF4 pathway is essential for stress-induced autophagy gene expression

In response to different environmental stresses, eIF2α phosphorylation represses global translation coincident with preferential translation of ATF4, a master regulator controlling the transcription of key genes essential for adaptative functions. Here, we establish that the eIF2α/ATF4 pathway directs an autophagy gene transcriptional program in response to amino acid starvation or endoplasmic reticulum stress. The eIF2α-kinases GCN2 and PERK and the transcription factors ATF4 and CHOP are also required to increase the transcription of a set of genes implicated in the formation, elongation and function of the autophagosome. We also identify three classes of autophagy genes according to their dependence on ATF4 and CHOP and the binding of these factors to specific promoter cis elements. Furthermore, different combinations of CHOP and ATF4 bindings to target promoters allow the trigger of a differential transcriptional response according to the stress intensity. Overall, this study reveals a novel regulatory role of the eIF2α–ATF4 pathway in the fine-tuning of the autophagy gene transcription program in response to stresses.

Overcoming NS1-Mediated Immune Antagonism Involves Both Interferon-Dependent and Independent Mechanisms

To ensure survival, our immune system must overcome the action of pathogen-encoded immune antagonists, such as influenza A nonstructural protein-1 (NS1). NS1 subverts the host interferon (IFN) response at multiple levels and blocks the induction of IFN-β, a critical antiviral cytokine. This immune antagonism can be overcome in some cases. It has been shown that IFN-β is upregulated by 48 h in the lungs of wild-type C57BL/6 mice infected with influenza A. In contrast, it is shown here that IFNB1 continues to be repressed in IFNAR1(-/-) IL28Rα(-/-) mice, which are deficient in Type-I and III IFN signaling, implying induction of IFNB1 depends on effective IFN signaling. Despite the complete lack of IFN signaling in this system, some IFN stimulated genes (ISGs) were induced following infection with a Flu strain lacking NS1. While the expression of these viral stress-inducible genes (VSIGs) was initially repressed following infection with wild-type Flu, many of these genes became upregulated by 48 h postinfection. These results demonstrate the existence of IFN-independent mechanisms that can overcome NS1-mediated immune antagonism of VSIGs.

Characterization of the first tuber mustard calmodulin-like gene, BjAAR1, and its functions in responses to abiotic stress and abscisic acid in Arabidopsis

The first tuber mustard calmodulin-like (CML) gene BjAAR1 (Brassica juncea var. tumida Tsen et Lee Abiotic stress and Abscisic acid (ABA) Responsive gene 1) was cloned and characterized. The protein encoded by BjAAR1 contains four predicted Ca2+ binding sites (EF-hand motif) and its recombinant protein can bind Ca2+ in vitro. qRT-PCR showed that the expression level of BjAAR1 was rather high in non-swollen stem of tuber mustard and largely reduced in swollen stem. Expression of BjAAR1 enhanced ABA- and stress-induced gene expression in Arabidopsis (Arabidopsis thaliana). Transgenic plants also exhibited hypersensitivity to NaCl, mannitol, and ABA during the seed germination and post-germination stages. ABA biosynthesis inhibitor, norflurazon (NF), rescued hypersensitivity phenotype of transgenic plants to NaCl and mannitol, indicating that BjAAR1 functions in multiple abiotic stresses response through ABA-dependent process.

Using natural variation in Drosophila to discover previously unknown endoplasmic reticulum stress genes

Natural genetic variation is a rich resource for identifying novel elements of cellular pathways such as endoplasmic reticulum (ER) stress. ER stress occurs when misfolded proteins accumulate in the ER and cells respond with the conserved unfolded protein response (UPR), which includes large-scale gene expression changes. Although ER stress can be a cause or a modifying factor of human disease, little is known of the amount of variation in the response to ER stress and the genes contributing to such variation. To study natural variation in ER stress response in a model system, we measured the survival time in response to tunicamycin-induced ER stress in flies from 114 lines from the sequenced Drosophila Genetic Reference Panel of wild-derived inbred strains. These lines showed high heterogeneity in survival time under ER stress conditions. To identify the genes that may be driving this phenotypic variation, we profiled ER stress-induced gene expression and performed an association study. Microarray analysis identified variation in transcript levels of numerous known and previously unknown ER stress-responsive genes. Survival time was significantly associated with polymorphisms in candidate genes with known (i.e., Xbp1) and unknown roles in ER stress. Functional testing found that 17 of 25 tested candidate genes from the association study have putative roles in ER stress. In both approaches, one-third of ER stress genes had human orthologs that contribute to human disease. This study establishes Drosophila as a useful model for studying variation in ER stress and identifying ER stress genes that may contribute to human disease.

Abstract 131: Alphavbeta3 Integrins Mediate Shear Stress-Induced Inflammation and Early Atherogenesis

Rational The frictional shear stress generated by blood flow activates integrins on the endothelial cell surface, and signaling downstream of newly activated integrins regulates several flow-induced responses including inflammation and permeability. Signaling through transitional matrix binding integrins (α5β1, αvβ3 and αvβ5) drives shear stress-induced NF-κB activation and proinflammatory gene expression; however the specific contribution of each of these integrins remains unclear. Objective To address which transitional matrix-binding integrin mediates shear stress-induced endothelial cell. Methods and Results We now show that the small molecule αv integrin inhibitor S247, but not the α5 inhibitor ATN-161, abolished flow-induced proinflammatory signaling (PAK, NF-κB). Blocking αv expression using siRNA similarly prevents flow-induced activation of PAK and NF-κB, whereas siRNA-mediated knock-down of α5 did not. Furthermore, αv and β3 knockdown but not α5 and β5 knockdown inhibited flow-induced ICAM-1 and VCAM-1 expression (both mRNA and protein) following either short term (5 hr) laminar and long term oscillatory (24 hr) shear stress, indicating αvβ3 is the mediator for flow induced endothelial cell inflammation. In contrast, αv knockdown did not affect TNFα-induced ICAM-1 and VCAM-1 expression, suggesting the anti-inflammatory effect of αv inhibition is specific to shear stress. Integrin inhibition did not affect all shear stress responses, as neither α5 nor αv siRNA affected shear stress-induced extracellular-signal regulated kinase (ERK1/2) activation. Lastly, inhibiting αv integrins using the small molecule inhibitor S247 reduces atherosclerotic plaque formation in ApoE knockout mice. Conclusion Taken together, these studies suggest that αvβ3 integrins mediate shear stress-induced proinflammatory responses associated with the transitional matrix in vitro and early atherosclerotic plaque formation in vivo .

RNA-Seq reveals expression signatures of genes involved in oxygen transport, protein synthesis, folding, and degradation in response to heat stress in catfish

Temperature is one of the most prominent abiotic factors affecting ectotherms. Most fish species, as ectotherms, have extraordinary ability to deal with a wide range of temperature changes. While the molecular mechanism underlying temperature adaptation has long been of interest, it is still largely unexplored with fish. Understanding of the fundamental mechanisms conferring tolerance to temperature fluctuations is a topic of increasing interest as temperature may continue to rise as a result of global climate change. Catfish have a wide natural habitat and possess great plasticity in dealing with environmental variations in temperature. However, no studies have been conducted at the transcriptomic level to determine heat stress-induced gene expression. In the present study, we conducted an RNA-Seq analysis to identify heat stress-induced genes in catfish at the transcriptome level. Expression analysis identified a total of 2,260 differentially expressed genes with a cutoff of twofold change. qRT-PCR validation suggested the high reliability of the RNA-Seq results. Gene ontology, enrichment, and pathway analyses were conducted to gain insight into physiological and gene pathways. Specifically, genes involved in oxygen transport, protein folding and degradation, and metabolic process were highly induced, while general protein synthesis was dramatically repressed in response to the lethal temperature stress. This is the first RNA-Seq-based expression study in catfish in response to heat stress. The candidate genes identified should be valuable for further targeted studies on heat tolerance, thereby assisting the development of heat-tolerant catfish lines for aquaculture.

Reproductive Organ and Vascular Specific Promoter of the Rice Plasma Membrane Ca2+ATPase Mediates Environmental Stress Responses in Plants

BackgroundPlasma membrane Ca2+ATPase is a transport protein in the plasma membrane of cells and helps in removal of calcium (Ca2+) from the cell, hence regulating Ca2+ level within cells. Though plant Ca2+ATPases have been shown to be involved in plant stress responses but their promoter regions have not been well studied.ResultsThe 1478 bp promoter sequence of rice plasma membrane Ca2+ATPase contains cis-acting elements responsive to stresses and plant hormones. To identify the functional region, serial deletions of the promoter were fused with the GUS sequence and four constructs were obtained. These were differentially activated under NaCl, PEG cold, methyl viologen, abscisic acid and methyl jasmonate treatments. We demonstrated that the rice plasma membrane Ca2+ATPase promoter is responsible for vascular-specific and multiple stress-inducible gene expression. Only full-length promoter showed specific GUS expression under stress conditions in floral parts. High GUS activity was observed in roots with all the promoter constructs. The −1478 to −886 bp flanking region responded well upon treatment with salt and drought. Only the full-length promoter presented cold-induced GUS expression in leaves, while in shoots slight expression was observed for −1210 and −886 bp flanking region. The −1210 bp deletion significantly responded to exogenous methyl viologen and abscisic acid induction. The −1210 and −886 bp flanking region resulted in increased GUS activity in leaves under methyl jasmonate treatments, whereas in shoots the −886 bp and −519 bp deletion gave higher expression. Salicylic acid failed to induce GUS activities in leaves for all the constructs.ConclusionsThe rice plasma membrane Ca2+ATPase promoter is a reproductive organ-specific as well as vascular-specific. This promoter contains drought, salt, cold, methyl viologen, abscisic acid and methyl jasmonate related cis-elements, which regulated gene expression. Overall, the tissue-specificity and inducible nature of this promoter could grant wide applicability in plant biotechnology.

Arabidopsis heat shock factor HsfA1a directly senses heat stress, pH changes, and hydrogen peroxide via the engagement of redox state

Arabidopsis heat shock factor HsfA1a is present in a latent, monomeric state under normal conditions; its activation involves heat stress-induced trimerization, binding to heat shock element in target promoters, and the acquisition of transcriptional competence. HsfA1a is an important regulator for heat stress-induced gene expression and thermotolerance. However, it is not clear whether HsfA1a is directly activated by stress and the mechanisms of the stress signaling are poorly understood. We analyzed HsfA1a activation by trimerization and DNA-binding assays invitro and invivo in response to heat stress, low/high pH, and hydrogen peroxide treatments. Our results show that purified recombinant HsfA1a was activated by these stress treatments invitro. The same treatments also induced the binding to HSP18.2 and HSP70 promoters as examined by chromatin immunoprecipitation, and the HsfA1a DNA binding paralleled the mRNA expression of its target genes induced by different stresses. Stress-induced DNA-binding could be reversed, both invitro and invivo, by subsequent incubation with reducing agents (DTT, NADPH). These data suggest that HsfA1a can directly sense stress and become activated, and this process is dependent on the redox state. An N-terminal deletion of the amino acid residues from 48 to 74 negatively affected pH- and hydrogen peroxide-, but not heat-stress sensing.

Neuroanatomy of Stress Responses

The autonomic nervous system, especially the sympathetic nervous system, regulates immune responses, while cytokines produced in the immune system also affect neuronal activities. Stress-induced expression of immediate early genes, such as c-Fos in the brain, and the viral transneuronal labeling using pseudorabies virus make it possible to analyze the neurocircuitry of the stress-related central autonomic nervous system. Limbic systems (amygdala, lateral septum, infralimbic, insular, ventromedial temporal cortical regions), and several hypothalamic and brainstem nuclei have been identified as the central sites that regulate stress-induced sympathetic nervous activation. This review focuses on the involvement of the amygdala in the regulation of stress-induced sympathetic nervous responses. All amygdaloid subnuclei receive psychological information from other limbic regions, while the lateral and central subnuclei receive sensory and immune information from parabrachial nucleus and medical geniculate nucleus. Output to the hypothalamus mainly originates from the medial amygdala, while output to the bed nucleus of the stria terminalis originates from the central amygdala and the medial amygdala. Sex steroids such as estrogen and androgen can modulate the sympathetic nervous activity since their receptors are expressed in the medial amygdala.

New Insights into S-nitrosylation in Multiple Sclerosis

S-nitrosylation is a biologically relevant post-translational protein modification with signaling consequence. In eukaryotes, a large number of proteins have been identified as S-nitrosylation targets. Derangement in protein S-nitrosylation has been implicated in the pathogenesis of a number of different disease entities including Multiple Sclerosis (MS). A growing body of evidence has shown that Nitric oxide (NO) plays a critical role in MS. NO and other reactive nitrogen species (RNS) are involved in neuroinflammation and neurodegeneration in MS. Signaling by RNS is carried out mainly by S-nitrosylation of critical cysteine residues in targeted proteins. In recent years, newer roles in MS have been attributed to RNS. These roles relate to S-nitrosylation of cysteines in proteins which has emerged as a potential new paradigm in signal transduction and regulation of protein function. In the present review we discuss the evidence for the diverse roles of S-nitrosylation in MS, including nitrosative stress-induced gene expression in MS, and S-nitrosylation of transcription factors in MS. In addition, S-nitrosylation can be therapeutically used in MS. Recent studies providing evidence for SNO-based therapy strategy in the treatment of MS will also be discussed. Undoubtedly, new exciting results will contribute to the expanding area of MS research.

Genes encoding plant-specific class III peroxidases are responsible for increased cold tolerance of the brassinosteroid-insensitive 1 mutant.

We previously reported that one of the brassinosteroidinsensitive mutants, bri1-9, showed increased cold tolerance compared with both wild type and BRI1-overexpressing transgenic plants, despite its severe growth retardation. This increased tolerance in bri1-9 resulted from the constitutively high expression of stress-inducible genes under normal conditions. In this report, we focused on the genes encoding class III plant peroxidases (AtPrxs) because we found that, compared with wild type, bri1-9 plants contain higher levels of reactive oxygen species (ROS) that are not involved with the activation of NADPH oxidase and show an increased level of expression of a subset of genes encoding class III plant peroxidases. Treatment with a peroxidase inhibitor, salicylhydroxamic acid (SHAM), led to the reduction of cold resistance in bri1-9. Among 73 genes that encode AtPrxs in Arabidopsis, we selected four (AtPrx1, AtPrx22, AtPrx39, and AtPrx69) for further functional analyses in response to cold temperatures. T-DNA insertional knockout mutants showed increased sensitivity to cold stress as measured by leaf damage and ion leakage. In contrast, the overexpression of AtPrx22, AtPrx39, and AtPrx69 increased cold tolerance in the BRI1-GFP plants. Taken together, these results indicate that the appropriate expression of a particular subset of AtPrx genes and the resulting higher levels of ROS production are required for the cold tolerance.

Histone deacetylase inhibitors suppress mechanical stress-induced expression of RUNX-2 and ADAMTS-5 through the inhibition of the MAPK signaling pathway in cultured human chondrocytes

To investigate the inhibitory effects and the regulatory mechanisms of histone deacetylase (HDAC) inhibitors on mechanical stress-induced gene expression of runt-related transcription factor (RUNX)-2 and adisintegrin and metalloproteinase with thrombospondin motif (ADAMTS)-5 in human chondrocytes. Human chondrocytes were seeded in stretch chambers at a concentration of 5 × 10(4)cells/chamber. Cells were pre-incubated with or without HDAC inhibitors (MS-275 or trichostatin A; TSA) for 12h, followed by uniaxial cyclic tensile strain (CTS) (0.5Hz, 10% elongation), which was applied for 30 min using the ST-140-10 system (STREX, Osaka, Japan). Total RNA was extracted and the expression of RUNX-2, ADAMTS-5, matrix metalloproteinase (MMP)-3, and MMP-13 at the mRNA and protein levels were examined by real-time polymerase chain reaction (PCR) and immunocytochemistry, respectively. The activation of diverse mitogen-activated protein kinase (MAPK) pathways with or without HDAC inhibitors during CTS was examined by western blotting. HDAC inhibitors (TSA: 10 nM, MS-275: 100 nM) suppressed CTS-induced expression of RUNX-2, ADAMTS-5, and MMP-3 at both the mRNA and protein levels within 1h. CTS-induced activation of p38 MAPK (p38), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) MAPKs was downregulated by both HDAC inhibitors. The CTS-induced expression of RUNX-2 and ADAMTS-5 was suppressed by HDAC inhibitors via the inhibition of the MAPK pathway activation in human chondrocytes. The results of the current study suggested a novel therapeutic role for HDAC inhibitors against degenerative joint disease such as osteoarthritis.