Activation of ERK1/2, JNK and PKB by hydrogen peroxide in human SH-SY5Y neuroblastoma cells:role of ERK1/2 in H2O2-induced cell death

Reactive oxygen species including H2O2 activate an array of intracellular signalling cascades that are closely associated with cell death and cell survival pathways. The human neuroblastoma SH-SY5Y cell line is widely used as model cell system for studying neuronal cell death induced by oxidative stress. However, at present very little is known about the signalling pathways activated by H2O2 in SH-SY5Y cells. Therefore, in this study we have investigated the effect of H2(O2 on extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK) and protein kinase B (PKB) activation in undifferentiated and differentiated SH-SY5Y cells. H2O2 stimulated time and concentration increases in ERK1/2, JNK and PKB phosphorylation in undifferentiated and differentiated SH-SY5Y cells. No increases in p38 MAPK phosphorylation were observed following H2O2 treatment. The phosphatidylinositol 3-kinase (PI-3K) inhibitors wortmannin and LY 294002 ((2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) inhibited H2O2-induced increases in ERK1/2 and PKB phosphorylation. Furthermore, H2O2-mediated increases in ERK1/2 activation were sensitive to the MAPK kinase 1 (MEK1) inhibitor PD 98059 (2'-amino-3'-methoxyflavone), whereas JNK responses were blocked by the JNK inhibitor SP 600125 (anthra[1-9-cd]pyrazol-6(2H)-one). Treatment of SH-SY5Y cells with H2O2 (1 mM; 16 h) significantly increased the release of lactate dehydrogenase (LDH) into the culture medium indicative of a decrease in cell viability. Pre-treatment with wortmannin, SP 600125 or SB 203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; p38 MAPK inhibitor) had no effect on H2O2-induced LDH release from undifferentiated or differentiated SH-SY5Y cells. In contrast, PD 98059 and LY 294002 significantly decreased H2O2-induced cell death in both undifferentiated and differentiated SH-SY5Y cells. In conclusion, we have shown that H2O2 stimulates robust increases in ERK1/2, JNK and PKB in undifferentiated and differentiated SH-SY5Y cells. Furthermore, the data presented clearly suggest that inhibition of the ERK1/2 pathway protects SH-SY5Y cells from H2O2-induced cell death.

Significant alterations in peripheral blood lymphocyte subsets in patients with somatoform disorder

Objective: Previous studies have suggested that somatoform disorders (SFD) might be associated with changes in the function of the central and autonomic nervous systems. The aim of this study was to examine the possible immunological differences between SFD and healthy controls. Methods: Twenty-four patients with SFD and 13 healthy individuals completed the psychological questionnaires to assess symptom reporting [Symptom Checklist-90 Revised (SCL-90-R)] and to diagnose for SFD [Screening for Somatoform Symptoms scale (SOMS-scale)]. Participants also provided a blood sample taken in the morning, which was analysed with an automated cell counter to determine the number of leucocytes per μl and with flow cytometry to determine lymphocyte subsets. Results: With the exception of a higher T4/T8 ratio in the patient group, which was mainly because of lower CD8 counts, there were no significant differences in the absolute number of lymphocytes (subsets) between patients with SFD and healthy subjects. A positive correlation between B-lymphocyte subsets (CD19+CD22+, CD19+CD5+, CD19+CD3-) to all scales of the SCL-90-R, except somatisation, were found in SFD. Additionally, a positive correlation was found in SFD between CD14+CD16+ monocytes and somatisation (0.573) on the SCL-90-R scale. Conclusion: These data indicate that patients with SFD have an enhanced humoral immunity as shown by increased B-cell numbers and furthermore an elevated T4/T8 ratio because of lower CD8 suppressor cells. Further studies will be required to determine whether these alterations in lymphocyte subsets are directly involved in the pathophysiology of SFD. © 2007 Blackwell Munksgaard.

Antenatal corticosteroids impact the inflammatory rather than the antiangiogenic profile of women with preeclampsia

Circulating antiangiogenic factors and proinflammatory cytokines are implicated in the pathogenesis of preeclampsia. This study was performed to test the hypothesis that steroids modify the balance of inflammatory and proangiogenic and antiangiogenic factors that potentially contribute to the patient’s evolving clinical state. Seventy singleton women, admitted for antenatal corticosteroid treatment, were enrolled prospectively. The study group consisted of 45 hypertensive women: chronic hypertension (n=6), severe preeclampsia (n=32), and superimposed preeclampsia (n=7). Normotensive women with shortened cervix (<2.5 cm) served as controls (n=25). Maternal blood samples of preeclampsia cases were obtained before steroids and then serially up until delivery. A clinical severity score was designed to clinically monitor disease progression. Serum levels of angiogenic factors (soluble fms-like tyrosine kinase-1 [sFlt-1], placental growth factor [PlGF], soluble endoglin [sEng]), endothelin-1 (ET-1), and proinflammatory markers (IL-6, C-reactive protein [CRP]) were assessed before and after steroids. Soluble IL-2 receptor (sIL-2R) and total immunoglobulins (IgG) were measured as markers of T- and B-cell activation, respectively. Steroid treatment coincided with a transient improvement in clinical manifestations of preeclampsia. A significant decrease in IL-6 and CRP was observed although levels of sIL-2R and IgG remained unchanged. Antenatal corticosteroids did not influence the levels of angiogenic factors but ET-1 levels registered a short-lived increase poststeroids. Although a reduction in specific inflammatory mediators in response to antenatal steroids may account for the transient improvement in clinical signs of preeclampsia, inflammation is unlikely to be the major contributor to severe preeclampsia or useful for therapeutic targeting.

Antenatal corticosteroids impact the inflammatory rather than the antiangiogenic profile of women with preeclampsia

Circulating antiangiogenic factors and proinflammatory cytokines are implicated in the pathogenesis of preeclampsia. This study was performed to test the hypothesis that steroids modify the balance of inflammatory and proangiogenic and antiangiogenic factors that potentially contribute to the patient's evolving clinical state. Seventy singleton women, admitted for antenatal corticosteroid treatment, were enrolled prospectively. The study group consisted of 45 hypertensive women: chronic hypertension (n=6), severe preeclampsia (n=32), and superimposed preeclampsia (n=7). Normotensive women with shortened cervix (<2.5 cm) served as controls (n=25). Maternal blood samples of preeclampsia cases were obtained before steroids and then serially up until delivery. A clinical severity score was designed to clinically monitor disease progression. Serum levels of angiogenic factors (soluble fms-like tyrosine kinase-1 [sFlt-1], placental growth factor [PlGF], soluble endoglin [sEng]), endothelin-1 (ET-1), and proinflammatory markers (IL-6, C-reactive protein [CRP]) were assessed before and after steroids. Soluble IL-2 receptor (sIL-2R) and total immunoglobulins (IgG) were measured as markers of T- and B-cell activation, respectively. Steroid treatment coincided with a transient improvement in clinical manifestations of preeclampsia. A significant decrease in IL-6 and CRP was observed although levels of sIL-2R and IgG remained unchanged. Antenatal corticosteroids did not influence the levels of angiogenic factors but ET-1 levels registered a short-lived increase poststeroids. Although a reduction in specific inflammatory mediators in response to antenatal steroids may account for the transient improvement in clinical signs of preeclampsia, inflammation is unlikely to be the major contributor to severe preeclampsia or useful for therapeutic targeting. © 2014 American Heart Association, Inc.

Family resemblances? Ligand binding and activation of family A and B G-protein-coupled receptors

In April 2007, the Biochemical Society held a meeting to compare and contrast ligand binding and activation of Family A and B GPCRs (G-protein-coupled receptors). Being the largest class, Family A GPCRs usually receive the most attention, although a previous Biochemical Society meeting has focused on Family B GPCRs. The aim of the present meeting was to bring researchers of both families together in order to identify commonalities between the two. The present article introduces the proceedings of the meeting, briefly commenting on the focus of each of the following articles. ©The Authors.

Increased expression of the ubiquitin-proteasome pathway in murine myotubes by proteolysis-inducing factor (PIF) is associated with activation of the transcription factor NF-kappa B

Proteolysis-inducing factor (PIF), isolated from a cachexia-inducing murine tumour, has been shown to stimulate protein breakdown in C 2C12 myotubes. The effect was attenuated by the specific proteasome inhibitor lactacystin and there was an elevation of proteasome 'chymotrypsin-like' enzyme activity and expression of 205 proteasome α-subunits at concentrations of PIF between 2 and 16 nM. Higher concentrations of PIF had no effect. The action of PIF was attenuated by eicosapentaenoic acid (EPA) (50 μM). At a concentration of 4 nM, PIF induced a transient decrease in IκBα levels after 30 min incubation, while no effect was seen at 20 nM PIF. The level of IκBα, an NF-κB inhibitory protein, returned to normal after 60 min. Depletion of IκBα from the cytosol was not seen in myotubes pretreated with EPA, suggesting that the NF-κB/IκB complex was stabilised. At concentrations between 2 and 8 nM, PIF stimulated an increased nuclear migration of NF-κB, which was not seen in myotubes pretreated with EPA. The PIF-induced increase in chymotrypsin-like enzyme activity was also attenuated by the NF-κB inhibitor peptide SN50, suggesting that NF-κB may be involved in the PIF-induced increase in proteasome expression. The results further suggest that EPA may attenuate protein degradation induced by PIF, at least partly, by preventing NF-κB accumulation in the nucleus. © 2003 Cancer Research UK.

Macrophages and lymphocyte responiveness to mitogen

Purified B-cells fail to proliferate in response to the strong thymus-independent (TI) antigen Lipopolysaccharide (LPS) in the absence of macrophages (Corbel and Melchers, 1983). The fact that macrophages, or factors derived from them are required is supported by the inability of marginal zone B-cells in infants to respond to highly virulent strains of bacteria such as Neisseria meningitidis and Streptococcus pneumoniae (Timens, 1989). This may be due to the lack of CD21 expression on B-cells in infants which could associate with its co-receptor (C3d) on adjacent macrophages. It is not clear whether cell surface contacts and/or soluble products are involved in lymphocyte-macrophage interactions in response to certain antigens. This thesis describes the importance of the macrophage in lymphocyte responses to T-dependent (TD) and TI antigens. The major findings of this thesis were as follows: (1). Macrophages were essential for a full proliferative response to a range of T - and B-cell mitogens and TI-1 and TI-2 antigens, including Concanavalin A, LPS, Pokeweed mitogen (PWM), Dextran sulphate, Phytohaemagglutinin-P (PHA-P) and Poly[I][C]. (2). A ratio of 1 macrophage to 1000 lymphocytes was sufficient for the mitogens to exert their effects. (3). The optimal conditions were established for the activation of an oxidative burst in cells of the monocyte/macrophage lineage as measured by luminometry. The order of ability was OpZ >PMA/lonomycin >f-MLP >Con A >DS >PHA >Poly[I][C] >LPS >PWM. Responses were only substantial and protracted with OpZ and PMA. Peritoneal macrophages were the most responsive cells, whereas splenic and alveolar macrophages were significantly less active and no response could be elicited with Kupffer cells, thus demonstrating heterogeneity between macrophages. (4). Activated macrophages that were then fixed with paraformaldehyde were unable to restore mitogenic responsiveness, even with a ratio of 1 macrophage to 5 lymphocytes. (5). Although highly purified T- and B-cells could respond to mitogen provided live macrophages were present, maximum activation was only observed when all 3 cell types were present. (6). Supernatants from purified macrophage cultures treated with a range of activators were able to partially restore lymphocyte responses to mitogen in macrophage-depleted splenocyte cultures, and purified T - and B-cell cultures. In fact supernatants from macrophages treated with LPS for only 30 minutes could restore responsiveness. Supernatants from OpZ treated macrophages were without effect. (7). Macrophage supernatants could not induce proliferation in the absence of mitogen. They therefore provide a co-mitogenic signal required by lymphocytes in order to respond to mitogen. (8). Macrophage product profiles revealed that LPS and Con A-treated macrophage supernatants showed elevated levels of IL-1β, TNF -α L TB4 and TXB2. These products were therefore good candidates as the co-mitogenic factor. The possible inhibitory factors secreted by OpZ-treated macrophages were PGE2, IL-10 and NO. (9). The removal of cytokines, eicosanoids and TNF-α from LPS-treated macrophage supernatants using Cycloheximide, Dexamethasone and an MMPI respectively, resulted in the inability of these supernatants to restore macrophage-depleted lymphocyte responses to mitogen. (10). rIL-1β and rTNF-α are co-mitogenic factors, as macrophage-depleted lymphocytes incubated with rIL-1β and rTNF-α can respond to mitogen.

Induction of proteasome expression in skeletal muscle is attenuated by inhibitors of NF-κB activation

The potential for inhibitors of nuclear factor-κB (NF-κB) activation to act as inhibitors of muscle protein degradation in cancer cachexia has been evaluated both in vitro and in vivo. Activation of NF-κB is important in the induction of proteasome expression and protein degradation by the tumour factor, proteolysis-inducing factor (PIF), since the cell permeable NF-κB inhibitor SN50 (18 μM) attenuated the expression of 205 proteasome α-subunits, two subunits of the 195 regulator MSSI and p42, and the ubiquitin-conjugating enzyme, E214k, as well as the decrease in myosin expression in murine myotubes. To assess the potential therapeutic benefit of NF-κB inhibitors on muscle atrophy in cancer cachexia, two potential inhibitors were employed; curcumin (50 μM) and resveratrol (30 μM). Both agents completely attenuated total protein degradation in murine myotubes at all concentrations of PIF, and attenuated the PIF-induced increase in expression of the ubiquitin-proteasome proteolytic pathway, as determined by the 'chymotrypsin-like' enzyme activity, proteasome subunits and E2 14k. However, curcumin (150 and 300 mg kg-1) was ineffective in preventing weight loss and muscle protein degradation in mice bearing the MAC16 tumour, whereas resveratrol (1 mg kg-1) significantly attenuated weight loss and protein degradation in skeletal muscle, and produced a significant reduction in NF-κB DNA-binding activity. The inactivity of curcumin was probably due to a low bioavailability. These results suggest that agents which inhibit nuclear translocation of NF-κB may prove useful for the treatment of muscle wasting in cancer cachexia.

Activation of protein kinase B by adenosine A1 and A3 receptors in newborn rat cardiomyocytes

It is well established that adenosine receptors are involved in cardioprotection and that protein kinase B (PKB) is associated with cell survival. Therefore, in this study we have investigated whether adenosine receptors (A1, A2A and A3) activate PKB by Western blotting and determined the involvement of phosphatidylinositol 3-kinase (PI-3K)/PKB in adenosine-induced preconditioning in cultured newborn rat cardiomyocytes. Adenosine (non-selective agonist), CPA (A1 selective agonist) and Cl-IB-MECA (A(3) selective agonist) all increased PKB phosphorylation in a time- and concentration-dependent manner. The combined maximal response to CPA and Cl-IB-MECA was similar to the increase in PKB phosphorylation induced by adenosine alone. CGS 21680 (A2A selective agonist) did not stimulate an increase in PKB phosphorylation. Adenosine, CPA and Cl-IB-MECA-mediated PKB phosphorylation were inhibited by pertussis toxin (PTX blocks G(i)/G(o)-protein), genistein (tyrosine kinase inhibitor), PP2 (Src tyrosine kinase inhibitor) and by the epidermal growth factor (EGF) receptor tyrosine kinase inhibitor AG 1478. The PI-3K inhibitors wortmannin and LY 294002 blocked A(1) and A(3) receptor-mediated PKB phosphorylation. The role of PI-3K/PKB in adenosine-induced preconditioning was assessed by monitoring Caspase 3 activity and lactate dehydrogenase (LDH) release induced by exposure of cardiomyocytes to 4 h hypoxia (0.5% O2) followed by 18 h reoxygenation (HX4/R). Pre-treatment with wortmannin had no significant effect on the ability of adenosine-induced preconditioning to reduce the release of LDH or Caspase 3 activation following HX4/R. In conclusion, we have shown for the first time that adenosine A1 and A3 receptors trigger increases in PKB phosphorylation in rat cardiomyocytes via a G1/G0-protein and tyrosine kinase-dependent pathway. However, the PI-3K/PKB pathway does not appear to be involved in adenosine-induced cardioprotection by preconditioning Adenosine A1 receptor .