Jun N-terminal kinase 1 regulates epithelial-to-mesenchymal transition induced by TGF-beta1

Transforming growth factor beta1 (TGF-beta1) is a cardinal cytokine in the pathogenesis of airway remodeling, and promotes epithelial-to-mesenchymal transition (EMT). As a molecular interaction between TGF-beta1 and Jun N-terminal kinase (JNK) has been demonstrated, the goal of this study was to elucidate whether JNK plays a role in TGF-beta1-induced EMT. Primary cultures of mouse tracheal epithelial cells (MTEC) from wild-type, JNK1-/- or JNK2-/- mice were comparatively evaluated for their ability to undergo EMT in response to TGF-beta1. Wild-type MTEC exposed to TGF-beta1 demonstrated a prominent induction of mesenchymal mediators and a loss of epithelial markers, in conjunction with a loss of trans-epithelial resistance (TER). Significantly, TGF-beta1-mediated EMT was markedly blunted in epithelial cells lacking JNK1, while JNK2-/- MTEC underwent EMT in response to TGF-beta1 in a similar way to wild-type cells. Although Smad2/3 phosphorylation and nuclear localization of Smad4 were similar in JNK1-/- MTEC in response to TGF-beta1, Smad DNA-binding activity was diminished. Gene expression profiling demonstrated a global suppression of TGF-beta1-modulated genes, including regulators of EMT in JNK1-/- MTEC, in comparison with wild-type cells. In aggregate, these results illuminate the novel role of airway epithelial-dependent JNK1 activation in EMT

Effectiveness of life skills training on increasing self-esteem of high school students

AbstractObjective This study designed to investigate effectiveness of training life skills on adolescents’ students. Method This study is a pseudo-experimental study which accomplished on 160 students in Karaj city. Subjects of the study selected randomly from list of students in all of the schools of Karaj; then they divided randomly in two groups. Trained counsellors taught the life skills to students of the study group, and 80 reminder subjects assigned as control group. After educating the training program, subjects administered Cooper Smith self-esteem questionnaire (58-items version). Results Findings of the study indicated that life skills training lead to significant increase of self-esteem in study group in contrast to control group subjects. Conclusion Psycho education and mental health programs such as life skills training could cause to increase the necessary skills in students and decline school and educational problems

Attenuation of lung fibrosis in mice with a clinically relevant inhibitor of glutathione-S-transferase π

Idiopathic pulmonary fibrosis (IPF) is a debilitating lung disease characterized by excessive collagen production and fibrogenesis. Apoptosis in lung epithelial cells is critical in IPF pathogenesis, as heightened loss of these cells promotes fibroblast activation and remodeling. Changes in glutathione redox status have been reported in IPF patients. S-glutathionylation, the conjugation of glutathione to reactive cysteines, is catalyzed in part by glutathione-S-transferase π (GSTP). To date, no published information exists linking GSTP and IPF to our knowledge. We hypothesized that GSTP mediates lung fibrogenesis in part through FAS S-glutathionylation, a critical event in epithelial cell apoptosis. Our results demonstrate that GSTP immunoreactivity is increased in the lungs of IPF patients, notably within type II epithelial cells. The FAS-GSTP interaction was also increased in IPF lungs. Bleomycin- and AdTGFβ-induced increases in collagen content, α-SMA, FAS S-glutathionylation, and total protein S-glutathionylation were strongly attenuated in Gstp(–/–) mice. Oropharyngeal administration of the GSTP inhibitor, TLK117, at a time when fibrosis was already apparent, attenuated bleomycin- and AdTGFβ-induced remodeling, α-SMA, caspase activation, FAS S-glutathionylation, and total protein S-glutathionylation. GSTP is an important driver of protein S-glutathionylation and lung fibrosis, and GSTP inhibition via the airways may be a novel therapeutic strategy for the treatment of IPF

Glutathione-S-transferase P promotes glycolysis in asthma in association with oxidation of pyruvate kinase M2

Background: Interleukin-1-dependent increases in glycolysis promote allergic airways disease in mice and disruption of pyruvate kinase M2 (PKM2) activity is critical herein. Glutathione-S-transferase P (GSTP) has been implicated in asthma pathogenesis and regulates the oxidation state of proteins via S-glutathionylation. We addressed whether GSTP-dependent S-glutathionylation promotes allergic airways disease by promoting glycolytic reprogramming and whether it involves the disruption of PKM2. Methods: We used house dust mite (HDM) or interleukin-1β in C57BL6/NJ WT or mice that lack GSTP. Airway basal cells were stimulated with interleukin-1β and the selective GSTP inhibitor, TLK199. GSTP and PKM2 were evaluated in sputum samples of asthmatics and healthy controls and incorporated analysis of the U-BIOPRED severe asthma cohort database. Results: Ablation of Gstp decreased total S-glutathionylation and attenuated HDM-induced allergic airways disease and interleukin-1β-mediated inflammation. Gstp deletion or inhibition by TLK199 decreased the interleukin-1β-stimulated secretion of pro-inflammatory mediators and lactate by epithelial cells. 13C-glucose metabolomics showed decreased glycolysis flux at the pyruvate kinase step in response to TLK199. GSTP and PKM2 levels were increased in BAL of HDM-exposed mice as well as in sputum of asthmatics compared to controls. Sputum proteomics and transcriptomics revealed strong correlations between GSTP, PKM2, and the glycolysis pathway in asthma. Conclusions: GSTP contributes to the pathogenesis of allergic airways disease in association with enhanced glycolysis and oxidative disruption of PKM2. Our findings also suggest a PKM2-GSTP-glycolysis signature in asthma that is associated with severe disease

Differential Requirement for c-Jun N-terminal Kinase 1 in Lung Inflammation and Host Defense

The c-Jun N-terminal kinase (JNK) - 1 pathway has been implicated in the cellular response to stress in many tissues and models. JNK1 is known to play a role in a variety of signaling cascades, including those involved in lung disease pathogenesis. Recently, a role for JNK1 signaling in immune cell function has emerged. The goal of the present study was to determine the role of JNK1 in host defense against both bacterial and viral pneumonia, as well as the impact of JNK1 signaling on IL-17 mediated immunity. Wild type (WT) and JNK1 −/− mice were challenged with Escherichia coli, Staphylococcus aureus, or Influenza A. In addition, WT and JNK1 −/− mice and epithelial cells were stimulated with IL-17A. The impact of JNK1 deletion on pathogen clearance, inflammation, and histopathology was assessed. JNK1 was required for clearance of E. coli, inflammatory cell recruitment, and cytokine production. Interestingly, JNK1 deletion had only a small impact on the host response to S. aureus. JNK1 −/− mice had decreased Influenza A burden in viral pneumonia, yet displayed worsened morbidity. Finally, JNK1 was required for IL-17A mediated induction of inflammatory cytokines and antimicrobial peptides both in epithelial cells and the lung. These data identify JNK1 as an important signaling molecule in host defense and demonstrate a pathogen specific role in disease. Manipulation of the JNK1 pathway may represent a novel therapeutic target in pneumonia

Validation of Binary Typing for Staphylococcus aureus Strains

Most of the DNA-based methods for genetic typing of Staphylococcus aureus strains generate complex banding patterns. Therefore, we have developed a binary typing procedure involving strain-differentiating DNA probes which were generated on the basis of randomly amplified polymorphic DNA (RAPD) analysis. We present and validate the usefulness of 15 DNA probes, according to generally accepted performance criteria for molecular typing systems. RAPD analysis with multiple primers was performed on 376 S. aureus strains of which 97% were methicillin resistant (MRSA). Among the 1,128 RAPD patterns generated, 66 were selected which identified 124 unique DNA fragments. From these amplicons, only 12% turned out to be useful for isolate-specific binary typing. The nature of the RAPD-generated DNA fragments was investigated by partial DNA sequence analysis. Several homologies with known S. aureus sequences and with genes from other species were discovered; however, 87% of the probe sequences are of previously unknown origin. The locations of most of the DNA probes on the chromosome of S. aureus NCTC 8325 were determined by hybridization. Seven fragments were randomly dispersed along the genome, five were clustered within the 2500- to 2600-kb position of the genome, and the remaining four did not recognize complementary sequences in S. aureus NCTC 8325. A total of 103 S. aureus strains (69% MRSA) were used for the validation of the binary typing technique. The 15 DNA probes provided stable epidemiological markers, both in vitro (type consistency after serial passages on culture media) and in vivo (comparison of sequential isolates recovered from cases of persistent colonization). The discriminatory power of binary typing (D = 0.998) exceeded that of pulsed-field gel electrophoresis (D = 0.966) and RAPD analysis (D = 0.949). Reproducibility, measured by analyzing multiple strains belonging to a multitude of different epidemiological clusters, was comparable to that of other genotyping techniques used. Contribution of the DNA probes to the discriminatory power of the system was analyzed by comparison of dendrograms. This study demonstrates that binary typing is a robust tool for the genetic typing of S. aureus isolates

Chronic Heart Failure Reduces Akt Phosphorylation in Human Skeletal Muscle: Relationship to Muscle Size and Function

HEART FAILURE (HF) is the final common pathway for many chronic cardiac diseases and is presently the only cardiac diagnosis continuing to increase in prevalence in the United States. Patients suffering from HF report high rates of physical disability, as defined by an inability to perform simple daily activities (43). Although the reason for their physical disability is unclear, most research has focused on aerobic exercise intolerance in these patients (23). This is logical considering that the hallmark symptom of HF is exertional dyspnea and because of the widespread use of aerobic capacity as a diagnostic tool (36). Diminished aerobic capacity, however, does not greatly limit the ability of patients to perform most daily activities (42). Instead, performance of many activities of daily living is strongly dependent on muscle strength (1, 4, 44), which is determined by the size of the muscle and its contractile properties. HF patients often experience muscle atrophy and weakness during the course of the disease, which may contribute to their physical disability. The mechanisms whereby HF alters skeletal muscle size and function, however, have not been clearly defined. The mass and function of skeletal muscle are largely dictated by its protein expression, which is determined by opposing anabolic and catabolic stimuli. Insulin-like growth factor-1 (IGF-1) is believed to be a key regulator of protein metabolism, stimulating anabolic (45) and inhibiting catabolic (51) pathways. One of the key mechanisms whereby IGF-1 mediates these effects downstream of receptor activation is through phosphorylation and activation of Akt (3). The anabolic effects of Akt are mediated, in part, through activation of mammalian target of rapamycin (mTOR) (37) and inhibition of glycogen synthase kinase-3β (GSK-3β) (9). Activation of mTOR stimulates protein translation through its effects on p70 ribosomal S6 kinase (p70 S6K) and eukaryotic translation initiation factor 4E binding protein-1 (eIF4E-BP) (15), whereas Akt-mediated phosphorylation of GSK-3β (9) stimulates protein translation (45) by diminishing its inhibitory phosphorylation of eIF2B (59). Additionally, Akt activation reduces protein breakdown via phosphorylation of forkhead box O (FOXO) transcription factors (46). In their phosphorylated form, FOXOs are excluded from the nucleus, where they would otherwise stimulate transcription of E3 ubiquitin ligases important for muscle proteolysis (46, 51). Thus IGF-1, working through the activation of Akt, promotes muscle protein anabolism through reciprocal regulation of protein synthesis and breakdown. Early studies suggested that HF reduces circulating IGF-1 levels (41), but few studies have evaluated the potential effect of these alterations on skeletal muscle. In animal models of HF, reduced skeletal muscle expression of IGF-1 was found and was related to decreased muscle fiber size (48). Moreover, administration of growth hormone (10), which stimulates muscle IGF-1 expression, or muscle-specific transgenic overexpression of IGF-1 (47) inhibits muscle atrophy and improves contractile function. In human HF, skeletal muscle IGF-1 mRNA abundance (20, 53) and protein expression (20) are reduced and are correlated with decreased muscle size (20) and myofibrillar gene expression (53). Based on these results, one might expect corresponding downregulation of signaling pathways downstream of IGF-1 receptor activation. However, the one study that has examined these downstream signaling events in humans found no effect of HF (27). In addition to the paucity of knowledge about signaling distal to receptor activation, none of these studies have accounted for the fact that HF patients have low levels of physical activity (55). Muscle use positively regulates skeletal muscle IGF-1 expression and activation of downstream signaling molecules (22, 30). Thus it is unclear whether diminished local IGF-1 expression observed in prior studies (20, 48, 53) is due to HF or is a consequence of muscle disuse that accompanies the disease. Considering these caveats, the unique effect of the HF syndrome on these variables remains undefined. The conventional wisdom is that diminished circulating IGF-1 and muscle IGF-1 expression in HF patients (20, 41, 53) promote muscle atrophy and dysfunction, but virtually no studies have evaluated signaling pathways within skeletal muscle that would translate the effect of IGF-1 on muscle size and function. Thus the primary objective of the present study was to evaluate the effect of HF on skeletal muscle IGF-1 expression and the phosphorylation status of signaling molecules downstream of IGF receptor activation. The present series of experiments focused on examining the phosphorylation status of the Akt/mTOR and Akt/GSK-3β pathways since we recently found no effect of HF on FOXO-regulated genes, atrogin and MuRF1, or markers of skeletal muscle protein breakdown (39). To examine the unique effects of HF, we recruited sedentary healthy controls to match patients for activity level and studied HF patients at least 6 mo following any hospitalization to minimize any influence of disease exacerbation or muscle disuse associated with bed rest. We hypothesized that HF would be characterized by reduced skeletal muscle IGF-1 expression and, correspondingly, altered phosphorylation of Akt, mTOR, GSK-3β, and downstream signaling molecules. Moreover, decreased IGF-1 expression and signaling molecule phosphorylation would be correlated to reduced muscle size and function