Telencephalin protects PAJU cells from amyloid beta protein-induced apoptosis by activating the ezrin/radixin/moesin protein family/phosphatidylinositol-3-kinase/protein kinase B pathway

Telencephalin is a neural glycoprotein that reduces apoptosis induced by amyloid beta protein in the human neural tumor cell line PAJU. In this study, we examined the role of the ezrin/radixin/moesin protein family/phosphatidylinositol-3-kinase/protein kinase B pathway in this process. Western blot analysis demonstrated that telencephalin, phosphorylated ezrin/radixin/moesin and phosphatidylinositol-3-kinase/protein kinase B were not expressed in PAJU cells transfected with empty plasmid, while they were expressed in PAJU cells transfected with a telencephalin expression plasmid. After treatment with 1.0 nM amyloid beta protein 42, expression of telencephalin and phosphorylated phosphatidylinositol-3-kinase/protein kinase B in the transfected cells gradually diminished, while levels of phosphorylated ezrin/radixin/moesin increased. In addition, the high levels of telencephalin, phosphorylated ezrin/radixin/moesin and phosphatidylinositol-3-kinase/protein kinase B expression in PAJU cells transfected with a telencephalin expression plasmid could be suppressed by the phosphatidylinositol-3-kinase inhibitor LY294002. These findings indicate that telencephalin activates the ezrin/radixin/moesin family/phosphatidylinositol-3-kinase/protein kinase B pathway and protects PAJU cells from amyloid beta protein-induced apoptosis.

Effect of G_(αq/11) Protein and ATP-sensitive Potassium Channels on Ischemic Preconditioning in Rat Hearts

Objectives To investigate the effect of Gαq/11 signaling pathway and ATP-sensitive potassium channel ( KATP channel ) on ischemic preconditioning (IPC) protection in rat hearts. Methods Two series of experiments were performed in Wistar rat hearts. In the first series of experiment, ischemic preconditioning was induced by left anterior descending occlusion (three, 5 min episodes separated by 5 min of reperfusion), ischemia-reperfusion injury was induced by 30 min coronary artery occlusion followed by 90 min reperfusion. Hemodynamics, infarct size and scores of ventricular arrhythmias were measured. The expression of Gαq/11 protein in the heart was measured by Western blot analysis in the second series. Results Ischemic preconditioning rats showed decreased infarct size and scores of ventricular arrhythmia vs non-IP control rats. The effect of IPC was significantly attenuated by glibenclamide (1 mg/kg, ip), a nonselective KATP channel inhibitor. IPC caused a significant increase in the expression of Gαq/11 protein. Conclusions Activations of Gαq/11 signal pathway and KATP channel played significant roles in the classical cardioprotection of ischemic precon-ditioning rat heart and might be an important mechanism of signal transduction pathway during the ischemic preconditioning.

Impact of rheumatoid arthritis-associated HLA-DRβ1 subtypes on protein kinase A signaling

Objective To investigate the impact of rheumatoid arthritis (RA)-associated HLA-DRβ1*0401, *0402, *0403, *0404 and *0101 subtypes on the protein kinase A (PKA) signaling pathway. Methods Adenylate cyclase (AC), cAMP and PKA activity in transfectants expressing RA-associated HLA-DRβ1 subtypes and their mutants were detected. Results Compared to HLA-DRβ1*0402 transfectants, the RA-associated HLA-DRβ1*0401, *0404 and *0101 transfectants produced significantly lower levels of AC, cAMP and PKA. Conclusion RA-associated HLA-DRβ1 molecules are involved in the pathogenesis of RA through down-regulation of the PKA signaling pathway.

Activation of mitogen activated protein kinases via complement receptor type 2

Background Complement receptor type 2 (CR2) is the receptor for C3d and C3dg and for Epstein Barr virus The aim of our study was to explore whether CR2 can independently mediate the activation of mitogen activated protein kinases (MAPKs, including ERK, JNK, and p38MAPK), and to highlight the molecular mechanism of CD 4 + cell deletion in AIDS Methods HOS cells (HOS CR2) and HOS CD4 cells (HOS CD4CR2) stably expressing CR2 were established and then identified by FACS and Western blotting Activation and blocking tests of MAPKs were assessed by Western blot Cell proliferation was determined using Cell Titer 96 Aqueous One Solution Reagent Results FACS results showed that the positive rates of HOS CR2 and HOS CD4CR2 cells were greater than 96%, and Western blot showed that the CR2 expression levels on HOS CR2 and HOS CD4CR2 cells were high Activation and blocking tests of MAPKs (ERK, JNK, and p38MAPK) were carried out in HOS CR2, HOS CD4, and HOS CD4CR2 cells The activation of MAPKs in HOS CR2 cells stimulated with PMA (100 ng/ml) and NHS (10%) was identical The activation of MAPKs increased at 5 minutes, reached a peak at 10 minutes, and decreased to baseline within 30 minutes, all in a time dependent manner; the activation of MAPKs was blocked by anti CR2 McAb, PD98059 (inhibitor of ERK), and Wortmanin (inhibitor of PI 3K), respectively In HOS CD4 cells, MAPKs were activated by HIV gp160 In HOS CD4CR2 cells, MAPK activation was induced by HIV gp160, 10% NHS, and HIV gp160+10%NHS; phosphorylation of p38MAPK was dramatically induced by HIV gp160+NHS, and lasted for 1 hour The cell proliferation results showed that HIV gp160 inhibited the proliferation of HOS CD4 and HOS CD4CR2 cells ( P <0 01) and that NHS enhanced the effect of HIV gp160 ( P <0 01) Conclusions The activation of MAPKs is independently mediated by CR2 and that anti CR2 McAb, PD98059, and Wortmanin block the activation of MAPKs, respectively The results of the signal transduction and cell proliferation assays of HOS CD4CR2 cells show that CR2 plays a role in the pathogenesis of HIV infection, especially in the inhibition of CD 4 + cell proliferation

G protein b_1λ_2 subunits purification and their interaction with adenylyl cyclase

A preliminary study on the interaction of G protein (guanine triphosphate binding pro- tein) b1g2 subunits and their coupled components in cell signal transduction was conducted in vitro. The insect cell lines, Sf9 (Spodoptera frugiperda) and H5 (Trichoplusia ni ) were used to express the recombinant protein Gb1g2. The cell membrane containing Gb1g2 was isolated through affinity chromatography column with Ni-NTA agarose by FPLC method, and the highly purified protein was obtained. The adenylyl cyclase 2 (AC2) activity assay showed that the purified Gb1g2 could signifi-cantly stimulate AC2 activity. The interaction of b1g2 subunits of G protein with the cytoplasmic tail of various mammalian adenylyl cyclases was monitored by BIAcore technology using NTA sensor chip, which relies on the phenomenon of surface plasmon resonance (SPR). The experiments showed the direct binding of Gb1g2 to the cytoplasmic tail C2 domain of AC2. The specific binding domain of AC2 with Gb1g2 was the same as AC2 activity domain which was stimulated by Gb1g2.

Primate Torpor: Regulation of Stress-activated Protein Kinases During Daily Torpor in the Gray Mouse Lemur, Microcebus murinus

Very few selected species of primates are known to be capable of entering torpor. This exciting discovery means that the ability to enter a natural state of dormancy is an ancestral trait among primates and, in phylogenetic terms, is very close to the human lineage. To explore the regulatory mechanisms that underlie primate torpor, we analyzed signal transduction cascades to discover those involved in coordinating tissue responses during torpor. The responses of mitogen-activated protein kinase（MAPK） family members to primate torpor were compared in six organs of control（aroused） versus torpid gray mouse lemurs, Microcebus murinus. The proteins examined include extracellular signal-regulated kinases（ERKs）, c-jun NH2-terminal kinases（JNKs）, MAPK kinase（MEK）, and p38, in addition to stress-related proteins p53 and heat shock protein 27（HSP27）. The activation of specific MAPK signal transduction pathways may provide a mechanism to regulate the expression of torpor-responsive genes or the regulation of selected downstream cellular processes. In response to torpor, each MAPK subfamily responded differently during torpor and each showed organ-specific patterns of response. For example, skeletal muscle displayed elevated relative phosphorylation of ERK1/2 during torpor. Interestingly, adipose tissues showed the highest degree of MAPK activation. Brown adipose tissue displayed an activation of ERK1/2 and p38, whereas white adipose tissue showed activation of ERK1/2, p38, MEK, and JNK during torpor. Importantly, both adipose tissues possess specialized functions that are critical for torpor, with brown adipose required for non-shivering thermogenesis and white adipose utilized as the primary source of lipid fuel for torpor. Overall, these data indicate crucial roles of MAPKs in the regulation of primate organs during torpor.

Angiotensin Ⅱ induced upregulation of Gαq/11,phospholipase Cβ_3 and extracellular signal-regulated kinase 1/2 via angiotensin Ⅱtype 1 receptor

Background The role of the Gαq/11-mediated signal transduction pathway in angiotensin Ⅱ (Ang Ⅱ) induced cardiac hypertrophy remains unclear. This study was to investigate the role of the Gαq/11 signal transduction pathway in the development of cardiac hypertrophy in 2K1C hypertensive rats and in cultured neonatal rat ventricular myocytes (NRVMs) and to elucidate the effects of the pathway on Ang Ⅱ induced cardiac hypertrophy. Methods Renal hypertension was induced in 2K1C hypertensive rats by placing a silver clip around the left renal artery. At 8 weeks after operation,the systolic blood pressure,the ratio of left ventricular weight to body weight (LV/BW),and the concentration of AngⅡ in the heart were measured. The protein levels of Gαq/11 and extracellular signal-regulated kinase 1/2 (ERK1/2) were assayed by Western blot analysis,and the activity of phospholipase C (PLC) in the myocardium was detected using [ 3H]-PIP_2 as a substrate. Changes in [ 3H]-leucine incorporation and in the protein levels of the signal molecules Gαq/11,PLCβ_3,and ERK1/2 were measured after NRVMs were stimulated with 10 -7 mol/L AngⅡ. Results The protein levels of Gαq/11 and ERK1/2 in the hearts of 2K1C rats increased by 35.8% and 31.9%,respectively,compared with the sham group. The PLC activity in the 2K1C group was also significantly increased ( P <0.05). The levels of Gαq/11,PLCβ_3,and ERK1/2 increased significantly after NRVMs were stimulated by AngⅡ. The upregulation of Gαq/11,PLCβ_3 and ERK1/2 in NRVMs occurred prior to [ 3H]-leucine incorporation increases,and could be inhibited with losartan. Conclusion AngⅡ can initiate cardiac hypertrophy and upregulate signal molecules in the Gαq/11-mediated signal transduction pathway,such as Gαq/11,PLCβ_3 and ERK1/2,at both tissue and cellular levels.