Up-regulation of IAPs by PI-3K: a cell survival signal-mediated anti-apoptotic mechanism

Under normal cell physiology, a balance between cell survival and apoptosis is crucial for homeostasis. Many studies have demonstrated that apoptosis is modulated by cell survival stimuli. Active Akt, a common mediator of cell survival signals, has been shown to inhibit apoptosis by attenuating activity of pro-apoptotic factors Bad and caspase-9. However, the anti-apoptotic mechanisms mediated by various cell survival signals are poorly understood. Human prostate cancer LNCaP cells, known to contain constitutively activated Akt as a result of a frame-shift mutation in PTEN, an inhibitor of PI-3K/Akt pathway, were observed to be completely resistant to TRAIL-induced apoptosis. In agreement with the known action of Akt, blockade of the PI-3K/Akt pathway rendered LNCaP cells highly susceptible to TRAIL. Importantly, active PI-3K/Akt prevented processing/activation of caspase-3, a phenomenon associated with the function of inhibitor of apoptosis proteins (IAPs). In fact, inhibition of PI-3K activity using Wortmannin significantly decreased the protein levels of IAPs, concomitantly promoting processing/activation of caspase-3 and TRAIL-induced apoptosis. My data indicate that in addition to blocking Bad and caspase-9 through Akt, PI-3K also inhibits caspase-3 through up-regulating IAPs, thereby attenuates apoptosis.     

Vanadate proliferative and anti-mineralogenic effects are mediated by MAPK and PI-3K/Ras/Erk pathways in a fish chondrocyte cell line

We recently reported proliferative and anti-mineralogenic effects of vanadate on fish chondrocytes and here we investigate the signalling pathways associated with these effects. Our data show that vanadate stimulates chondrocyte proliferation through the MAPK pathway, using signalling mechanisms similar to those used by IGF-1, while it inhibits chondrocyte differentiation/mineralization through a putative PI-3K/Ras/Erk signalling, a pathway shared with insulin. Our data also suggest that vanadate impairs ECM mineralization not only by interfering with regulatory pathways but also by inhibiting enzymatic activity of ALP. Finally, this work provides additional evidence for the conservation, throughout evolution, of mechanisms regulating chondrocyte proliferation and differentiation.     

Intracellular Ca(2+) and Ca(2+)/calmodulin-dependent kinase II mediate acute potentiation of neurotransmitter release by neurotrophin-3

Neurotrophins have been shown to acutely modulate synaptic transmission in a variety of systems, but the underlying signaling mechanisms remain unclear. Here we provide evidence for an unusual mechanism that mediates synaptic potentiation at the neuromuscular junction (NMJ) induced by neurotrophin-3 (NT3), using Xenopus nerve-muscle co-culture. Unlike brain-derived neurotrophic factor (BDNF), which requires Ca(2+) influx for its acute effect, NT3 rapidly enhances spontaneous transmitter release at the developing NMJ even when Ca(2+) influx is completely blocked, suggesting that the NT3 effect is independent of extracellular Ca(2+). Depletion of intracellular Ca(2+) stores, or blockade of inositol 1, 4, 5-trisphosphate (IP3) or ryanodine receptors, prevents the NT3-induced synaptic potentiation. Blockade of IP3 receptors can not prevent BDNF-induced potentiation, suggesting that BDNF and NT3 use different mechanisms to potentiate transmitter release. Inhibition of Ca(2+)/calmodulin-dependent kinase II (CaMKII) completely blocks the acute effect of NT3. Furthermore, the NT3-induced potentiation requires a continuous activation of CaMKII, because application of the CaMKII inhibitor KN62 reverses the previously established NT3 effect. Thus, NT3 potentiates neurotransmitter secretion by stimulating Ca(2+) release from intracellular stores through IP3 and/or ryanodine receptors, leading to an activation of CaMKII.     

Molecular dynamics simulations reveal structural insights into inhibitor binding modes and mechanism of casein kinase II inhibitors

Casein kinase-II, a member of protein kinase family, plays significant role in different cellular processes such as cell growth, differentiation, proliferation, gene expression, and embryogenesis. Being a potent suppressor of apoptosis, it serves as a significant link for its association with various types of malignancies such as colorectal and breast cancer. To overcome its pathological role in various cancerous diseases, CK-II procures great consideration as a therapeutic target. This study aimed at understanding the binding mechanism and structural properties of benzimidazole derivatives by utilizing various computational tools including docking simulation, three-dimensional quantitative structure activity relationships and molecular dynamic simulation. Structure-based 3D-QSAR techniques such as CoMFA and CoMSIA models, were established from the conformations gained by protein–ligand docking approach. The attained models have showed a good extrapolative power for internal as well as external validation. Moreover, MD simulation was carried out to explain the detailed binding mechanism and the comparison of inhibitor’s binding mode with diverse biological activities. A good correlation was observed among docking studies, MD results, and contour map analysis. Interestingly new molecules were designed using detail structural information from MD simulation, showed higher potency of inhibition (pIC₅₀ 7.6–7.7) compare to the most active compound of the series.     

A(2A) adenosine receptor facilitation of neuromuscular transmission: influence of stimulus paradigm on calcium mobilization

The influence of stimulus pulse duration on calcium mobilization triggering facilitation of evoked [(3)H]acetylcholine ([(3)H]ACh) release by the A(2A) adenosine receptor agonist CGS 21680C was studied in the rat phrenic nerve-hemidiaphragm. The P-type calcium channel blocker omega-agatoxin IVA (100 nM) decreased [(3)H]ACh release evoked with pulses of 0.04-ms duration, whereas nifedipine (1 microM) inhibited transmitter release with pulses of 1-ms duration. Depletion of intracellular calcium stores by thapsigargin (2 microM) decreased [(3)H]ACh release evoked by pulses of 1 ms, an effect observed even in the absence of extracellular calcium. With short (0.04-ms) stimulation pulses, when P-type calcium influx triggered transmitter release, facilitation of [(3)H]ACh release by CGS 21680C (3 nM) was attenuated by both thapsigargin (2 microM) and nifedipine (1 microM). With longer stimuli (1 ms), a situation in which both thapsigargin-sensitive internal stores and L-type channels are involved in ACh release, pretreatment with either omega-agatoxin IVA (100 nM) or nifedipine (1 microM) reduced the facilitatory effect of CGS 21680C (3 nM). The results suggest that A(2A) receptor activation facilitates ACh release from motor nerve endings through alternatively mobilizing the available calcium pools (thapsigargin-sensitive internal stores and/or P- or L-type channels) that are not committed to the release process in each stimulation condition.     

Autophagy induced by AXL receptor tyrosine kinase alleviates acute liver injury via inhibition of NLRP3 inflammasome activation in mice

Severe hepatic inflammation is a common cause of acute or chronic liver disease. Macrophages are one of the key mediators which regulate the progress of hepatic inflammation. Increasing evidence shows that the TAM (TYRO3, AXL and MERTK) family of RTKs (receptor tyrosine kinases), which is expressed in macrophages, alleviates inflammatory responses through a negative feedback loop. However, the functional contribution of each TAM family member to the progression of hepatic inflammation remains elusive. In this study, we explore the role of individual TAM family proteins during autophagy induction and evaluate their contribution to hepatic inflammation. Among the TAM family of RTKs, AXL (AXL receptor tyrosine kinase) only induces autophagy in macrophages after interaction with its ligand, GAS6 (growth arrest specific 6). Based on our results, autophosphorylation of 2 tyrosine residues (Tyr815 and Tyr860) in the cytoplasmic domain of AXL in mice is required for autophagy induction and AXL-mediated autophagy induction is dependent on MAPK (mitogen-activated protein kinase)14 activity. Furthermore, induction of AXL-mediated autophagy prevents CASP1 (caspase 1)-dependent IL1B (interleukin 1, β) and IL18 (interleukin 18) maturation by inhibiting NLRP3 (NLR family, pyrin domain containing 3) inflammasome activation. In agreement with these observations, axl^?/? mice show more severe symptoms than do wild-type (Axl^+/+) mice following acute hepatic injury induced by administration of lipopolysaccharide (LPS) or carbon tetrachloride (CCl₄). Hence, GAS6-AXL signaling-mediated autophagy induction in murine macrophages ameliorates hepatic inflammatory responses by inhibiting NLRP3 inflammasome activation.     

Angiotensin II receptor blocker telmisartan enhances running endurance of skeletal muscle through activation of the PPAR‐δ/AMPK pathway

Clinical trials have shown that angiotensin II receptor blockers reduce the new onset of diabetes in hypertensives; however, the underlying mechanisms remain unknown. We investigated the effects of telmisartan on peroxisome proliferator activated receptor γ (PPAR‐δ) and the adenosine monophosphate (AMP)‐activated protein kinase (AMPK) pathway in cultured myotubes, as well as on the running endurance of wild‐type and PPAR‐δ‐deficient mice. Administration of telmisartan up‐regulated levels of PPAR‐δ and phospho‐AMPKα in cultured myotubes. However, PPAR‐δ gene deficiency completely abolished the telmisartan effect on phospho‐AMPKαin vitro. Chronic administration of telmisartan remarkably prevented weight gain, enhanced running endurance and post‐exercise oxygen consumption, and increased slow‐twitch skeletal muscle fibres in wild‐type mice, but these effects were absent in PPAR‐δ‐deficient mice. The mechanism is involved in PPAR‐δ‐mediated stimulation of the AMPK pathway. Compared to the control mice, phospho‐AMPKα level in skeletal muscle was up‐regulated in mice treated with telmisartan. In contrast, phospho‐AMPKα expression in skeletal muscle was unchanged in PPAR‐δ‐deficient mice treated with telmisartan. These findings highlight the ability of telmisartan to improve skeletal muscle function, and they implicate PPAR‐δ as a potential therapeutic target for the prevention of type 2 diabetes.     

PTEN, a negative regulator of PI3 kinase signalling, alters tau phosphorylation in cells by mechanisms independent of GSK-3

Deregulation of PTEN/Akt signalling has been recently implicated in the pathogenesis of Alzheimer's disease (AD), but the effects on the molecular processes underlying AD pathology have not yet been fully described. Here we report that overexpression of PTEN reduces tau phosphorylation in CHO cells. This effect was abrogated by mutant PTEN constructs with either a catalytically inactive point mutation (C124S) or with only inactive lipid phosphatase activity (G129E), suggesting an indirect, lipid phosphatase-dependent process. The predominant effects of PTEN on tau appeared to be mediated by reducing ERK1/2 activity, but were independent of Akt, GSK-3, JNK and the tau phosphatases PP1 and PP2A. Our studies provide evidence for an effect of PTEN on the phosphorylation of tau in AD pathogenesis, and provide some insight into the mechanisms through which deregulation of PTEN may contribute towards the progression of tauopathy.     

The preparation of cyclic nucleotide-depleted regulatory subunits from type II adenosine 3':5'-monophosphate-dependent protein kinase by a nondenaturing method

A nondenaturing method for the preparation of R subunits from type II cyclic AMP-dependent protein kinase is described. The procedure is based on the exchange of cyclic AMP, which is tightly bound to the R subunit, for more weakly bound cyclic GMP, which can be removed by washing and dialysis. Less than 5% of the available cyclic nucleotide-binding sites of R subunit prepared by this method contained cyclic AMP and less than 3% contained cyclic GMP. The C-subunit contamination (mol of C/mol of R monomer) was approximately 0.2%. These levels of contamination did not affect the properties of the R subunit as judged by (a) the ability of the R subunit to inhibit the activity of the C subunit and (b) the rate of exchange of cAMP into R2 . etheno-cAMP. The advantages of our method are that the protein is not subjected to denaturing conditions and that large amounts of material can be processed relatively rapidly.     

Opposite effects of presynaptic 5-HT3 receptor activation on spontaneous and action potential-evoked GABA release at hippocampal synapses

5-HT(3) (serotonin type 3) receptors are targets of antiemetics, antipsychotics, and antidepressants and are believed to play a role in cognition. Nevertheless, contrasting results have been obtained with respect to their functions in the CNS and in the control of transmitter release. We used rat hippocampal neurons in single-neuron microcultures to identify the roles of presynaptic 5-HT(3) receptors at central synapses. 5-HT (10 microm) caused a transient > 10-fold increase in the frequency of miniature inhibitory postsynaptic currents without affecting amplitudes or kinetics. This effect was abolished by tropisetron (30 nm) and when Ca(2+) channels were blocked by 100 microm Cd(2+) it was mimicked and occluded when neurons were depolarized by 20 mm, but not 10 mm, K(+). Thus, activation of presynaptic 5-HT(3) receptors increased spontaneous GABA release by causing depolarization and opening of voltage-gated Ca(2+) channels. In microculture neurons, 5-HT transiently reduced action potential-evoked inhibitory autaptic currents by > 50%; this effect was blocked by tropisetron and mimicked by 20 mm, but not 10 mm, K(+). Miniature excitatory postsynaptic currents were not altered by 5-HT. Excitatory autaptic currents were tonically reduced, an effect attenuated by 5-HT(1A) antagonists. Thus, presynaptic 5-HT(3) receptors control GABA, but not glutamate, release and mediate opposite effects on spontaneous and action potential-dependent release.     

A quench-flow kinetic investigation of calcium ion accumulation by isolated cardiac sarcoplasmic reticulum. Dependence of initial velocity on free calcium ion concentration and influence of preincubation with a protein kinase,MgATP, and cyclic AMP

Ca²⁺ accumulation at pH 6.8 by isolated rabbit heart microsomes derived chiefly from sarcoplasmic reticulum was investigated by a quench-flow technique. The reaction was terminated at preset times by addition to the reaction mixture of an equal volume of 10 to 50 mM ethyleneglycol-bis-(β-aminoethyl ether)-N,N′-tetraacetic acid buffered at pH 6.0. The initial velocity of Ca²⁺ accumulation by microsomal preparations exhibiting a steady state Ca²⁺ accumulation of 25.6 nmol Ca²⁺/mg increased from 3.67 to 33.4 nmol Ca²⁺/mg · s as the free Ca²⁺ concentration was raised from 0.2 to 18.9 μM. Preincubation of the cardiac microsomes with a partly purified soluble cardiac cyclic AMP-dependent protein kinase, MgATP, and cyclic AMP lead to a significant increase in the initial Ca²⁺ accumulation rate. The amounts of Ca²⁺ that were found to accumulate in the first 200 ms of the reaction are comparable to the quantities of the ion that according to literature data need to be removed from the myofilaments and the myoplasm for induction of relaxation of the myocardial fibers.     

The protective effect of trefoil factor 3 on the intestinal tight junction barrier is mediated by toll-like receptor 2 via a PI3K/Akt dependent mechanism

Trefoil factor peptides are highly conserved secreted molecules characterized by heat and enzymatic digestion resistance. Intestinal trefoil factor 3 (TFF3) protects and repairs the gastrointestinal mucosa and restores normal intestinal permeability, which is dependent on the integrity of the tight junction (TJ) barrier and the TJ associated proteins claudin-1, zona occludens-1 (ZO-1) and occludin. Despite the important role of intestinal barrier dysfunction in the pathogenesis of inflammatory bowel diseases, the underlying mechanisms and associated molecules remain unclear. In the present study, we show that TFF3 and toll-like receptor 2 (TLR2) are functionally linked and modulate intestinal epithelial permeability via a mechanism that involves the PI3K/Akt pathway. We used the Caco-2 cell model to show that TLR2 and TFF3 inhibit the IL-1β induced increase in permeability and release of proinflammatory cytokines, and that this effect is mediated by activation of PI3K/Akt signaling. TLR2 silencing downregulated the expression of TFF3 and overexpression of TLR2 and TFF3 increased the levels of phospho-Akt. TFF3 overexpression significantly upregulated the expression of ZO-1, occludin and claudin-1 and this effect was abrogated by inhibition of the PI3K/Akt pathway. Taken together, our results indicate that TLR2 signaling selectively enhances intestinal TJ barrier integrity through a mechanism involving TFF3 and the activation of the PI3K/Akt pathway.     

Saikosaponin C inhibits lipopolysaccharide-induced apoptosis by suppressing caspase-3 activation and subsequent degradation of focal adhesion kinase in human umbilical vein endothelial cells

Bacterial lipopolysaccharide (LPS) is an important mediator of inflammation and a potent inducer of endothelial cell damage and apoptosis. In this study, we investigated the protective effects of saikosaponin C (SSc), one of the active ingredients produced by the traditional Chinese herb, Radix Bupleuri, against LPS-induced apoptosis in human umbilical endothelial cells (HUVECs). LPS triggered caspase-3 activation, which was found to be important in LPS-induced HUVEC apoptosis. Inhibition of caspase-3 also inhibited LPS-induced degradation of focal adhesion kinase (FAK), indicating that caspase-3 is important in LPS-mediated FAK degradation as well as in apoptosis in HUVECs. SSc significantly inhibited LPS-induced apoptotic cell death in HUVECs through the selective suppression of caspase-3. SSc was also shown to rescue LPS-induced FAK degradation and other cell adhesion signals. Furthermore, the protective effects of SSc against LPS-induced apoptosis were abolished upon pretreatment with a FAK inhibitor, highlighting the importance of FAK in SSc activity. Taken together, these results show that SSc efficiently inhibited LPS-induced apoptotic cell death via inhibition of caspase-3 activation and caspase-3-mediated-FAK degradation. Therefore, SSc represents a promising therapeutic candidate for the treatment of vascular endothelial cell injury and cellular dysfunction.     

The membrane effects of 17beta-estradiol on chondrocyte phenotypic expression are mediated by activation of protein kinase C through phospholipase C and G-proteins

Growth plate chondrocytes from both male and female rats have nuclear receptors for 17β-estradiol (E₂); however, recent studies indicate that an alternative pathway involving a membrane receptor may also be involved in the female cell response. E₂ directly affects the fluidity of chondrocyte membranes derived from female, but not male, rats. In addition, E₂ activates PKC in a nongenomic manner in female cells, and chelerythrine, a specific inhibitor of PKC, inhibits E₂-dependent alkaline phosphatase activity in these cells, indicating PKC is involved in the signal transduction mechanism. The aims of this study were: (1) to examine if PKC mediates the effect of E₂ on chondrocyte proliferation, differentiation, and matrix synthesis; and (2) to determine the pathway that mediates the membrane effect of E₂ on PKC. Confluent, fourth passage resting zone (RC) and growth zone (GC) chondrocytes from female rat costochondral cartilage were treated with 10⁻¹⁰ to 10⁻⁷ M E₂ in the presence or absence of the PKC inhibitor chelerythrine, and changes in alkaline phosphatase specific activity, proteoglycan sulfation, and [³H]thymidine incorporation were measured. To examine the pathway of PKC activation, chondrocyte cultures were treated with E₂ in the presence or absence of genistein (an inhibitor of tyrosine kinases), U73122 or D609 (inhibitors of phospholipase C [PLC]), quinacrine (an inhibitor of phospholipase A₂ [PLA₂]), and melittin (an activator of PLA₂). Alkaline phosphatase specific activity and proteoglycan sulfation were increased and [³H]thymidine incorporation was decreased by E₂. The effects of E₂ on all parameters were blocked by chelerythrine. Treatment of the cultures with E₂ produced a significant dose-dependent increase in PKC. U73122 dose-dependently inhibited the activation of PKC in E₂-stimulated female chondrocyte cultures. However, the classical receptor antagonist ICI 182780 was unable to block the stimulatory effect of E₂ on PKC. Moreover, the classical receptor agonist diethylstilbestrol (DES) had no effect on PKC, nor did it alter the stimulatory effect of E₂. Inhibition of tyrosine kinase and PLA₂ had no effect on the activation of PKC by E₂. The PLA₂ activator also had no effect on PKC activation by E₂. E₂ stimulated PKC activity in membranes isolated from the chondrocytes, demonstrating a direct membrane effect for this steroid hormone. These data indicate that the rapid nongenomic effect of E₂ on PKC activity in chondrocytes from female rats is sex-specific and dependent upon a G-protein-coupled phospholipase C.     

The membrane effects of 17β-estradiol on chondrocyte phenotypic expression are mediated by activation of protein kinase C through phospholipase C and G-proteins

Growth plate chondrocytes from both male and female rats have nuclear receptors for 17β-estradiol (E₂); however, recent studies indicate that an alternative pathway involving a membrane receptor may also be involved in the female cell response. E₂ directly affects the fluidity of chondrocyte membranes derived from female, but not male, rats. In addition, E₂ activates PKC in a nongenomic manner in female cells, and chelerythrine, a specific inhibitor of PKC, inhibits E₂-dependent alkaline phosphatase activity in these cells, indicating PKC is involved in the signal transduction mechanism. The aims of this study were: (1) to examine if PKC mediates the effect of E₂ on chondrocyte proliferation, differentiation, and matrix synthesis; and (2) to determine the pathway that mediates the membrane effect of E₂ on PKC. Confluent, fourth passage resting zone (RC) and growth zone (GC) chondrocytes from female rat costochondral cartilage were treated with 10⁻¹⁰ to 10⁻⁷ M E₂ in the presence or absence of the PKC inhibitor chelerythrine, and changes in alkaline phosphatase specific activity, proteoglycan sulfation, and [³H]thymidine incorporation were measured. To examine the pathway of PKC activation, chondrocyte cultures were treated with E₂ in the presence or absence of genistein (an inhibitor of tyrosine kinases), U73122 or D609 (inhibitors of phospholipase C [PLC]), quinacrine (an inhibitor of phospholipase A₂ [PLA₂]), and melittin (an activator of PLA₂). Alkaline phosphatase specific activity and proteoglycan sulfation were increased and [³H]thymidine incorporation was decreased by E₂. The effects of E₂ on all parameters were blocked by chelerythrine. Treatment of the cultures with E₂ produced a significant dose-dependent increase in PKC. U73122 dose-dependently inhibited the activation of PKC in E₂-stimulated female chondrocyte cultures. However, the classical receptor antagonist ICI 182780 was unable to block the stimulatory effect of E₂ on PKC. Moreover, the classical receptor agonist diethylstilbestrol (DES) had no effect on PKC, nor did it alter the stimulatory effect of E₂. Inhibition of tyrosine kinase and PLA₂ had no effect on the activation of PKC by E₂. The PLA₂ activator also had no effect on PKC activation by E₂. E₂ stimulated PKC activity in membranes isolated from the chondrocytes, demonstrating a direct membrane effect for this steroid hormone. These data indicate that the rapid nongenomic effect of E₂ on PKC activity in chondrocytes from female rats is sex-specific and dependent upon a G-protein-coupled phospholipase C.