Activated protein C promotes protease-activated receptor-1 cytoprotective signaling through β-arrestin and dishevelled-2 scaffolds

Protease-activated receptor-1 (PAR1) is a guanine nucleotide-binding (G) protein-coupled receptor that elicits cellular responses to coagulant and anticoagulant proteases. Activation of PAR1 by the coagulant protease thrombin results in Ras homolog gene family member A (RhoA) activation, disassembly of adherens junctions, and disruption of the endothelial barrier. In contrast, activation of PAR1 with the anticoagulant protease activated protein C (APC) results in activation of Ras-related C3 botulinum toxin substrate 1 (Rac1) and endothelial barrier protection. We previously showed that APC cytoprotective signaling requires the compartmentalization of PAR1 in caveolar microdomains. However, the mechanism by which APC-activated PAR1 promotes cytoprotective signaling in human endothelial cells remains poorly understood. Here we show that APC-activated PAR1 cytoprotective signaling is mediated by β-arrestin recruitment and activation of the dishevelled-2 (Dvl-2) scaffold and not by G protein α inhibiting activity polypeptide 2 (Gα(i)) signaling. In human endothelial cells, PAR1 and β-arrestins form a preassembled complex and cosegregate in caveolin-1-enriched fractions. Remarkably, we found that depletion of β-arrestin expression by RNA interference resulted in the loss of APC-induced Rac1 activation but not of thrombin-stimulated RhoA signaling. APC also failed to protect against thrombin-induced endothelial barrier permeability in cells deficient in β-arrestin expression. We further demonstrate that APC activation of PAR1 results in β-arrestin-dependent recruitment of Dvl-2, which is critical for Rac1 signaling and endothelial barrier protection but not for thrombin-induced RhoA signaling. Our findings identify a role for β-arrestin and Dvl-2 scaffolds in APC-activated PAR1 cytoprotective signaling in human endothelial cells.     

Angiotensin II upregulates protein phosphatase 2Cα and inhibits AMP-activated protein kinase signaling and energy balance leading to skeletal muscle wasting

Congestive heart failure and chronic kidney disease are characterized by chronically elevated angiotensin II (Ang II) and muscle wasting. Ang II causes skeletal muscle wasting by reducing appetite and by enhancing catabolism. The serine/threonine kinase AMP-activated protein kinase (AMPK) functions mainly as a sensor of cellular energy status. It is energy sparing and favors ATP generation. We hypothesized that Ang II induces muscle wasting in part by inhibiting AMPK signaling and altering cellular energy balance. Our results show that Ang II infusion in mice reduced gastrocnemius muscle weight by 26% and depleted ATP by 74%. In addition, Ang II upregulated protein phosphatase 2Cα by 2.6-fold and reduced AMPK phosphorylation and signaling in muscle. Importantly, the pharmacological AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside restored AMPK activity to levels of pair-fed controls and reversed Ang II-mediated ATP depletion and muscle wasting. Moreover, 5-aminoimidazole-4-carboxamide ribonucleoside activated Akt and inhibited Ang II-induced increases in E3 ubiquitin ligase expression. These novel results demonstrate critical roles for energy depletion and AMPK inhibition in Ang II-induced skeletal muscle wasting and suggest a therapeutic potential for AMPK activators in diseases characterized by muscle wasting.     

Ubiquitin-like modifier activating enzyme 2 promotes cell migration and invasion through Wnt/β-catenin signaling in gastric cancer

AIM To investigate the function and mechanism of ubiquitinlike modifier activating enzyme 2(Uba2) in progression of gastric cancer(GC) cells.METHODS Uba2 level in patients with GC was analyzed by Western blotting and immunohistochemistry. MTT and colony formation assays were performed to examine cell proliferation.Flow cytometry was used for cell cycle analysis.Wound healing and Transwell assays were conducted to examine the effects of Uba2 on migration and invasion.Expression levels of cell cycle-related proteins, epithelial-mesenchymal transition(EMT) biomarkers, and involvement of the Wnt/β-catenin pathway was assessed by Western blotting. Activation of the Wnt/β-catenin pathway was confirmed by luciferase assay.RESULTS Uba2 expression was higher in GC than in normal tissues.Increased Uba2 expression was correlated with tissue differentiation, Lauren's classification, vascular invasion,and TNM stage, as determined by the analysis of 100 GC cases(P 0.05). Knock-down of Uba2 inhibited GC cell proliferation, induced cell cycle arrest, and altered expression of cyclin D1, P21, P27, and Bcl-2, while upregulation of Uba2 showed the opposite effects. The wound healing and Transwell assays showed that Uba2 promoted GC cell migration and invasion. Western blotting revealed alterations in EMT biomarkers, suggesting the role of Uba2 in EMT. Furthermore, the luciferase reporter assay indicated the involvement of the Wnt/β-catenin signaling pathway as a possible modulator of Uba2 oncogenic functions.CONCLUSION Uba2 plays a vital role in GC cell migration and invasion,possibly by regulating the Wnt/β-catenin signaling pathway and EMT.     

Post–conditioning induced cardioprotection requires signaling through a redox–sensitive mechanism, mitochondrial ATP–sensitive K+ channel and protein kinase C activation

Post–conditioning (Post–C) induced cardioprotection involves activation of guanylyl–cyclase. In the ischemic preconditioning scenario, the downstream targets of cGMP include mitochondrial ATP–sensitive K⁺ (mK_ATP) channels and protein kinase C (PKC), which involve reactive oxygen species (ROS) production. This study tests the hypothesis that mK_ATP, PKC and ROS are also involved in the Post–C protection. Isolated rat hearts underwent 30 min global ischemia (I) and 120 min reperfusion (R) with or without Post–C (i.e., 5 cycles of 10 s R/I immediately after the 30 min ischemia). In 6 groups (3 with and 3 without Post–C) either mK_ATP channel blocker, 5– hydroxydecanoate (5–HD), or PKC inhibitor, chelerythrine (CHE) or ROS scavenger, N–acetyl–cysteine (NAC), were given during the entire reperfusion (120 min). In other 6 groups (3 with and 3 without Post–C), 5–HD, CHE or NAC were infused for 117 min only starting after 3 min of reperfusion not to interfere with the early effects of Post–C and/or reperfusion. In an additional group NAC was given during Post–C maneuvers (i.e., 3 min only). Myocardial damage was evaluated using nitro–blue tetrazolium staining and lactate dehydrogenase (LDH) release. Post–C attenuated myocardial infarct size (21 ± 3% vs. 64 ± 5% in control; p < 0.01). Such an effect was abolished by 5–HD or CHE given during either the 120 or 117 min of reperfusion as well as by NAC given during the 120 min or the initial 3 min of reperfusion. However, delayed NAC (i.e., 117 min infusion) did not alter the protective effect of Post– C (infarct size 32 ± 5%; p < 0.01 vs. control, NS vs. Post–C). CHE, 5–HD or NAC given in the absence of Post–C did not alter the effects of I/R. Similar results were obtained in terms of LDH release. Our data show that Post–C induced protection involves an early redox–sensitive mechanism as well as a persistent activation of mK_ATP and PKC, suggesting that the mK_ATP/ROS/PKC pathway is involved in post–conditioning.     

T cell signaling: Protein kinase Cθ the immunological synapse and characterization of SLAT a novel T helper 2-specific adapter protein

Triggering of the antigen-specific T cell receptor (TCR) can lead to various functional outcomes, such as activation and proliferation, anergy or cell death. This differential signaling is mainly determined by the quality and quantity of TCR signals, the nature of accessory signals and the differentiation/maturation status of the T cell. In this regard, T cell development and differentiation of the two major T helper (Th) subsets, namely Th1 and Th2 cells, can also be viewed as examples of differential signaling. In the present report, we review two T cell-selective signaling molecules (protein kinase C (PKC) θ and SLAT), which we have studied extensively and that appear to play important roles in the process of differential signaling. The novel PKC isoform PKCθ is selectively expressed in T lymphocytes and is essential for TCR-triggered activation of mature T cells via activation of the nuclear factor-κB and activator protein-1 pathways. Productive engagement of T cells by antigen-presenting cells (APC) results in recruitment of PKCθ to the T cell-APC contact area, the immunological synapse (IS), where it interacts with several signaling molecules to induce activation signals essential for productive T cell activation and interleukin-2 production. These events are associated with PKCθ translocation to membrane lipid rafts, which also localize to the IS. The Vav/Rac pathway promotes the recruitment of PKCθ to the IS or lipid rafts as well as its activation. SLAT is a novel adapter protein, which we isolated recently. It is selectively expressed in Th2 lineage cells, where it is found associated with the TCR-coupled protein tyrosine kinase ZAP-70. Our initial characterization of SLAT indicates that, by regulating the overall strength of TCR signaling, it may play an important role in differential signaling processes, which promote the differentiation and activation of allergy promoting and anti-inflammatory Th2 cells.     

Ca(2+) /calmodulin- dependent protein kinase II mediates transforming growth factor-β-induced hepatic stellate cells proliferation but not in collagen α1(I) production

Aim: Hepatic stellate cells (HSC) are the major players in hepatic fibrosis. As a most potent mitogen, transforming growth factor-β (TGF-β) strongly activates HSC and increases intracellular Ca(2+) concentration. Here, we assessed the potential role of Ca(2+) /calmodulin-dependent protein kinase II (CaMKII), a main downstream effector of the Ca(2+) signal in liver fibrogenesis cascade. Methods: A human immortal HSC cell line, LX-2, and primary rat hepatic stellate cells were used in current study. CaMKII blockage and Akt inhibition were performed by KN-93/CaMKIIα siRNA and LY294002, respectively. HSC proliferation was detected by 5-bromodeoxyuridine incorporation assay. Real-time polymerase chain reaction, western blot and enzyme-linked immunosorbent assay were used to measure mRNA, cellular protein and protein in medium, respectively. Procollagen α1(I) expression was detected by immunocytochemistry. The role of CaMKII on TGF-β/Smad-induced collagen α1(I) expression was determined by (CAGA)(12) -MLP luciferase activity assay. Results: TGF-β dramatically increased CaMKII mRNA, and total and phosphorylated CaMKII expression. KN-93 and CaMKIIα siRNA suppressed TGF-β-mediated HSC proliferation. CaMKII interruption blocked TGF-β-elicited Akt activation. LY294002 arrested HSC proliferation and collagen α1(I) production but had no effect on CaMKII. Furthermore, CaMKII led to increased p21 and p27 expression. KN-93 and CaMKIIα siRNA inhibited TGF-β-induced and basal collagen α1(I) production but had no effect on the activity of (CAGA)(12) -MLP luciferase in response to TGF-β stimulation. Conclusion: CaMKII is a pivotal signal in TGF-β-induced fibrogenic cascades by means of stimulating HSC proliferation, and involved in a basal collagen production. Therefore, CaMKII will be a potentially effective target in the development of therapeutic intervention strategies to attenuate hepatic fibrosis.     

CD95 triggers Orai1-mediated localized Ca2+ entry, regulates recruitment of protein kinase C (PKC) β2, and prevents death-inducing signaling complex formation

The death receptor CD95 plays a pivotal role in immune surveillance and immune tolerance. Binding of CD95L to CD95 leads to recruitment of the adaptor protein Fas-associated death domain protein (FADD), which in turn aggregates caspase-8 and caspase-10. Efficient formation of the CD95/FADD/caspase complex, known as the death-inducing signaling complex (DISC), culminates in the induction of apoptosis. We show that cells exposed to CD95L undergo a reorganization of the plasma membrane in which the Ca(2+) release-activated Ca(2+) channel Orai1 and the endoplasmic reticulum-resident activator stromal interaction molecule 1 colocalize with CD95 into a micrometer-sized cluster in which the channel elicits a polarized entry of calcium. Orai1 knockdown and expression of a dominant negative construct (Orai1E106A) reveal that on CD95 engagement, the Orai1-driven localized Ca(2+) influx is fundamental to recruiting the Ca(2+)-dependent protein kinase C (PKC) β2 to the DISC. PKCβ2 in turn transiently holds the complex in an inactive status, preventing caspase activation and transmission of the apoptotic signal. This study identifies a biological role of Ca(2+) and the Orai1 channel that drives a transient negative feedback loop, introducing a lag phase in the early steps of the CD95 signal. We suggest that these localized events provide a time of decision to prevent accidental cell death.