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The integrin receptor protein talin plays vital roles in intracellular chemical and mechanical activities, and it is implicated in the high invasion and poor prognosis of non-small cell lung cancer (NSCLC). To better understand the mechanism underlying the function of talin in NSCLC invasion and metastasis, a few newly designed tension probe based on Förster resonance energy transfer was used for real-time observation of tension changes in A549 cells. High NSCLC cell aggressiveness was found to be accompanied with inward talin and outward glial fibrillary acidic protein (GFAP) tensions, which are closely associated with microfilament (MF) force and intracellular osmotic potential. The increased osmotic pressure resulted from the production of intracellular protein nanoparticles and the related ion influx. Furthermore, integrin activation was found to adjust the talin and GFAP tensions. Disruption of the interaction between talin and MFs blocked the mechanical source of talin, reducing both talin tension and osmotic pressure and thus inhibiting NSCLC cell invasion and migration. Consequently, our study demonstrates that talin is involved in NSCLC invasion and migration via its inward tension and that the integrin pathway is correlated closely with protein-nanoparticle-induced outward osmotic pressure.

Aims Macrophages, as an important member of immune system, engulf and digest pathogens in innate immunity and help initiate adaptive immunity. However, macrophages also involve in occurrence and development of many diseases, such as obesity and type 2 diabetes. Here, we aimed to reveal how activated macrophages cause insulin resistance in skeletal muscle in vitro through simulating body environment. Main methods We established RAW264.7 macrophages and C2C12 myotubes co-incubation model in vitro using Transwell filter to simulate body environment and investigated effects of RAW264.7 cells on insulin-regulated glucose metabolism in C2C12 myotubes. Immunofluorescence, Immunoblot and glucose uptake tests were used to assess metabolic changes in C2C12 myotubes. ELISA test detected secretions of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) from RAW264.7 cells. In addition, RNA interference and inhibitor treatment were used. Key findings Activated RAW264.7 cells attenuated insulin response in C2C12 myotubes. Activated RAW264.7 cells secreted a lot of TNF-α and IL-6. We found that TNFα, but not IL-6, caused insulin resistance of skeletal muscle in a dose-dependent manner. The results further indicated that activation of TNF-α downstream proteins, inhibitor of nuclear factor κ-B kinase (IKK) and the jun-N-terminal kinase 1 (JNK1) led to phosphorylation of insulin receptor substrate 1 (IRS-1) at Ser residues and insulin resistance in C2C12 myotubes. Significance Our research provided further and direct demonstration on activated macrophage-induced insulin resistance in skeletal muscle, suggesting TNF-α might become a therapeutic target to ameliorate and treat type 2 diabetes.

The allosteric coupling of the highly conserved nucleotide- and substrate-binding domains of Hsp70 has been studied intensively. In contrast, the role of the disordered, highly variable C-terminal region of Hsp70 remains unclear. In many eukaryotic Hsp70s, the extreme C-terminal EEVD motif binds to the tetratricopeptide-repeat domains of Hsp70 co-chaperones. Here, we discovered that the TVEEVD sequence of Saccharomyces cerevisiae cytoplasmic Hsp70 (Ssa1) functions as a SUMO-interacting motif. A second C-terminal motif of ∼15 amino acids between the α-helical lid and the extreme C terminus, previously identified in bacterial and eukaryotic organellar Hsp70s, is known to enhance chaperone function by transiently interacting with folding clients. Using structural analysis, interaction studies, fibril formation assays, and in vivo functional assays, we investigated the individual contributions of the α-helical bundle and the C-terminal disordered region of Ssa1 in the inhibition of fibril formation of the prion protein Ure2. Our results revealed that although the α-helical bundle of the Ssa1 substrate-binding domain (SBDα) does not directly bind to Ure2, the SBDα enhances the ability of Hsp70 to inhibit fibril formation. We found that a 20-residue C-terminal motif in Ssa1, containing GGAP and GGAP-like tetrapeptide repeats, can directly bind to Ure2, the Hsp40 co-chaperone Ydj1, and α-synuclein, but not to the SUMO-like protein SMT3 or BSA. Deletion or substitution of the Ssa1 GGAP motif impaired yeast cell tolerance to temperature and cell-wall damage stress. This study highlights that the C-terminal GGAP motif of Hsp70 is important for substrate recognition and mediation of the heat shock response.

Translationally controlled tumor protein (TCTP) is an abundant protein that is highly conserved in eukaryotes. However, its primary function is still not clear. Human TCTP interacts with the metazoan-specific eukaryotic elongation factor 1Bδ (eEF1Bδ) and inhibits its guanine nucleotide exchange factor (GEF) activity, but the structural mechanism remains unknown. The interaction between TCTP and eEF1Bδ was investigated by NMR titration, structure determination, paramagnetic relaxation enhancement, site-directed mutagenesis, isothermal titration calorimetry, and HADDOCK docking. We first demonstrated that the catalytic GEF domain of eEF1Bδ is not responsible for binding to TCTP but rather a previously unnoticed central acidic region (CAR) domain in eEF1Bδ. The mutagenesis data and the structural model of the TCTP-eEF1Bδ CAR domain complex revealed the key binding residues. These residues are highly conserved in eukaryotic TCTPs and in eEF1B GEFs, including the eukaryotically conserved eEF1Bα, implying the interaction may be conserved in all eukaryotes. Interactions were confirmed between TCTP and the eEF1Bα CAR domain for human, fission yeast, and unicellular photosynthetic microalgal proteins, suggesting that involvement in protein translation through the conserved interaction with eEF1B represents a primary function of TCTP.

Double-stranded RNA-dependent protein kinase (PKR), an intracellular pathogen recognition receptor, is involved both in insulin resistance in peripheral tissues and in downregulation of pancreatic β-cell function in a kinase-dependent manner, indicating PKR as a core component in the progression of type 2 diabetes. PKR also acts as an adaptor protein via its protein-binding domain. Here, the PKR protein-binding function promoted β-cell proliferation without its kinase activity, which is associated with enhanced physical interaction with tumor necrosis factor receptor-associated factor 2 (TRAF2) and TRAF6. In addition, the transcription of the nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB)-dependent survival gene c-Myc was upregulated significantly and is necessary for proliferation. Upregulation of the PKR protein-binding function induced the NF-κB pathway, as observed by dose-dependent degradation of IκBα, induced nuclear translocation of p65 and elevated NF-κB-dependent reporter gene expression. NF-κB-dependent reporter activity and β-cell proliferation both were suppressed by TRAF2-siRNA, but not by TRAF6-siRNA. TRAF2-siRNA blocked the ubiquitination of receptor-interacting serine/threonine-protein kinase 1 (RIP1) induced by PKR protein binding. Furthermore, RIP1-siRNA inhibited β-cell proliferation. Proinflammatory cytokines (TNFα) and glucolipitoxicity also promoted the physical interaction of PKR with TRAF2. Collectively, these data indicate a pivotal role for PKR’s protein-binding function on the proliferation of pancreatic β cells through TRAF2/RIP1/NF-κB/c-Myc pathways. Therapeutic opportunities for type 2 diabetes may arise when its kinase catalytic function, but not its protein-binding function, is downregulated.

Hsp70 is a highly conserved chaperone that in addition to providing essential cellular functions and aiding in cell survival following exposure to a variety of stresses is also a key modulator of prion propagation. Hsp70 is composed of a nucleotide-binding domain (NBD) and substrate-binding domain (SBD). The key functions of Hsp70 are tightly regulated through an allosteric communication network that coordinates ATPase activity with substrate-binding activity. How Hsp70 conformational changes relate to functional change that results in heat shock and prion-related phenotypes is poorly understood. Here, we utilised the yeast [PSI +] system, coupled with SBD-targeted mutagenesis, to investigate how allosteric changes within key structural regions of the Hsp70 SBD result in functional changes in the protein that translate to phenotypic defects in prion propagation and ability to grow at elevated temperatures. We find that variants mutated within the β6 and β7 region of the SBD are defective in prion propagation and heat-shock phenotypes, due to conformational changes within the SBD. Structural analysis of the mutants identifies a potential NBD:SBD interface and key residues that may play important roles in signal transduction between domains. As a consequence of disrupting the β6/β7 region and the SBD overall, Hsp70 exhibits a variety of functional changes including dysregulation of ATPase activity, reduction in ability to refold proteins and changes to interaction affinity with specific co-chaperones and protein substrates. Our findings relate specific structural changes in Hsp70 to specific changes in functional properties that underpin important phenotypic changes in vivo. A thorough understanding of the molecular mechanisms of Hsp70 regulation and how specific modifications result in phenotypic change is essential for the development of new drugs targeting Hsp70 for therapeutic purposes.

Double-stranded RNA-dependent protein kinase (PKR) is revealed to participate in the development of insulin resistance in peripheral tissues in type 2 diabetes (T2DM). Meanwhile, PKR is also characterized as a critical regulator of cell proliferation. To date, no study has focused on the impact of PKR on the proliferation of pancreatic β-cells. Here, we adopted insulinoma cell lines and mice islet β-cells to investigate: (1) the effects of glucolipotoxicity and pro-inflammatory cytokines on PKR activation; (2) the effects of PKR on proliferation of pancreatic β-cells and its underlying mechanisms; (3) the actions of PKR on pro-proliferative effects of IGF-I and its underlying pathway. Our results provided the first evidence that PKR can be activated by glucolipitoxicity and pro-inflammatory cytokines in pancreatic β-cells, and activated PKR significantly inhibited cell proliferation by arresting cell cycle at G1 phase. Reductions in cyclin D1 and D2 as well as increases in p27 and p53 were associated with the anti-proliferative effects of PKR, and proteasome-dependent degradation took part in the reduction of cyclin D1 and D2. Besides, PKR activation abrogated the pro-proliferative effects of IGF-I by activating JNK and disrupting IRS1/PI3K/Akt signaling pathway. These findings indicate that the anti-proliferative actions of PKR on pancreatic β-cells may contribute to the pathogenesis of T2DM.

Arsenic trioxide (ATO) has shown anticancer activity against a variety of solid tumor models through induction of apoptosis, promotion of cellular differentiation, and inhibition of cellular invasive ability. The present study investigated the role of ceramide in regulating the invasive activity of hepatoma carcinoma HCCLM3 cells during ATO treatment. We found that ATO treatment inhibited HCCLM3 cell invasion and downregulated matrix metalloproteinase-9 (MMP-9) protein levels in a concentration-dependent manner. ATO also dose dependently induced the generation and accumulation of ceramide in HCCLM3 cells. Blockage of intracellular ceramide production through the inhibition of de novo ceramide synthesis or the hydrolysis of sphingomyelin increased the invasive ability and upregulated MMP-9 protein levels. The findings of this study indicated that ATO induced ceramide production through de novo ceramide synthesis and the hydrolysis of sphingomyelin and suggested that ceramide accumulation in response to ATO stimuli may play an important role in cancer therapy.

Background Ceramide accumulation is considered a contributing factor to neuronal dysfunction and damage. However, the underlying mechanisms that occur following ischemic insult are still unclear. Methods In the present study, we established cerebral ischemia models using four-vessel occlusion and oxygen-glucose deprivation methods. The hippocampus neural cells were subjected to immunohistochemistry and immunofluorescence staining for ceramide and neutral sphingomyelinase 2 (nSMase2) levels; immunoprecipitation and immunoblot analysis for nSMase2, receptor for activated C kinase 1 (RACK1), embryonic ectoderm development (EED), p38 mitogen-activated protein kinase (p38MAPK) and phosphorylated p38MAPK expression; SMase assay for nSMase and acid sphingomyelinase (aSMase) activity; real-time reverse transcription polymerase chain reaction for cytokine expression; and Nissl, microtubule-associated protein 2 and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling staining. Results We found considerable production of ceramide in astrocytes, but not in neurons, during early cerebral ischemia. This was accompanied by the induction of nSMase (but not aSMase) activity in the rat hippocampi. The inhibition of nSMase2 activity effectively reduced ceramide accumulation in astrocytes and alleviated neuronal damage to some extent. Meanwhile, the expression levels of proinflammatory cytokines, including tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β) and IL-6, were found to be upregulated, which may have played an import role in neuronal damage mediated by the nSMase2/ceramide pathway. Although enhanced binding of nSMase2 with RACK1 and EED were also observed after cerebral ischemia, nSMase2 activity was not blocked by the TNF-α receptor inhibitor through RACK1/EED signaling. p38MAPK, but not protein kinase Cζ or protein phosphatase 2B, was able to induce nSMase2 activation after ischemia. p38MAPK can be induced by A2B adenosine receptor (A2BAR) activity. Conclusions These results indicate that the inhibition of ceramide production in astrocytes by targeting A2BAR/p38MAPK/nSMase2 signaling may represent a viable approach for attenuating inflammatory responses and neuronal damage after cerebral ischemia.

Background The inactivation of c-Jun N-terminal kinase (JNK) is associated with anti-apoptotic and anti-inflammatory effects in cerebral ischemia, which can be induced by an imbalance between upstream phosphatases and kinases. Result Mitogen-activated protein kinase phosphatase 7 (MKP-7) was upregulated significantly at 4 h of reperfusion postischemia in rat hippocampi. By administration of cycloheximide or siRNA against mitogen-activated protein kinase phosphatase 7 (MKP-7) in a rat model of ischemia/reperfusion, an obvious enhancement of JNK activity was observed in 4 h of reperfusion following ischemia, suggesting MKP-7 was involved in JNK inactivation after ischemia. The subcellular localization of MKP-7 altered after ischemia, and the inhibition of MKP-7 nuclear export by Leptomycin B up-regulated JNK activity. Although PI3K/Akt inhibition could block downregulation of JNK activity through SEK1 and MKK-7 activation, PI3K/Akt activity was not associated with the regulation of JNK by MKP-7. Conclusions MKP-7, independently of PI3K/Akt pathway, played a key role in downregulation of JNK activity after ischemia in the rat hippocampus, and the export of MKP-7 from the nucleus was involved in downregulation of cytoplasmic JNK activity in response to ischemic stimuli.

Extracellular-signal-regulated kinase (ERK) undergoes rapid inactivation following the intense activation evoked by cerebral ischemia and reperfusion. However, the precise mechanism of this inactivation has not been elucidated. To investigate how phosphatases regulate the ERK cascade following ischemia, the PP2A inhibitors cantharidin and okadaic acid were administrated to the CA1 subregion of the rat hippocampus. The resulting sustained ERK activity implies that PP2A is a major phosphatase contributing to the rapid inactivation, but not activation, of ERK following cerebral ischemia. The increase in PP2A activity induced by ceramide has a weak effect on the activation of Raf via dephosphorylation of Ser259 in response to ischemia. In contrast, ketamine (Keta) and cyclosporine A (CsA), two chemicals that block calcium signal in ischemia, decrease ERK activity by blocking Raf dephosphorylation of Ser259. We also observed that activation of an upstream protein, Ras-GRF, leads to calcium/calmodulin-dependent activation of the ERK signaling cascade in response to ischemic stimuli. In addition, the activity of cyclic AMP response element-binding protein (CREB) and estrogen receptor α (ERα), target proteins of ERK and protective elements against ischemic lesion, parallels the activity of ERK. These data indicate that PP2A plays a significant role in blocking the protective effect induced by the ERK kinase pathway and that fast inactivation of ERK is the result of cross talk between calcium/calmodulin-dependent, positively regulated signal cascades and a ceramide-dependent negative signaling pathway. © 2008 Wiley-Liss, Inc.

Production of reactive oxygen species (ROS) results in up-regulation of the extracellular signal-regulated kinase (ERK) cascade in response to numerous stimuli. Cerebral ischemia induces calcium-dependent kinase activation followed by ROS production. Here, we examined how ROS mediates the activation of ERK following cerebral ischemia in the rat hippocampus. We found that alpha-tocopherol, a free radical scavenger, attenuated the initial, robust activation of ERK by inhibiting Raf-1 dephosphorylation at Ser259. Alpha-tocopherol also down-regulated the second and mild activation of ERK through inhibition of Src-dependent phosphorylation of Raf-1 at Tyr340/341. Our results suggest that ROS production mediates the biphasic activation of ERK through different signaling cascades following post-ischemic reperfusion. Mediation of these signaling pathways involves changes in Raf-1 phosphorylation at different sites.

Extracellular signal-regulated kinases (ERKs) can be activated by calcium signals. In this study, we investigated whether calcium-dependent kinases were involved in ERKs cascade activation after global cerebral ischemia.Cerebral ischemia was induced by four-vessel occlusion, and the calcium-dependent proteins were detected by immunoblot.Lethal-simulated ischemia significantly resulted in ERKs activation in N-methyl-D-aspartate (NMDA) receptor-dependent manner, accompanying with differential upregulation of Src kinase and Ca2+/calmodulin-dependent protein kinase II (CaMKII) activities. With the inhibition of Src family tyrosine kinases or CaMKII by administration of PP2 or KN62, the phosphorylation of ERKs was impaired dramatically during post-ischemia recovery. However, ischemic challenge also repressed ERKs activity when Src kinase was excessively activated.Src family tyrosine kinases and CaMKII might be involved in the activation of ERKs mediated by NMDA receptor in response to acute ischemic stimuli in vivo, but the intense activation of Src kinase resulted from ischemia may play a reverse role in the ERKs cascade.