The Key Role Of Protein Kinase D Specifically Mediates Apoptosis Signal-regulating Kinase 1-JNK Signaling Induced By H₂O₂ And Its Mechanism Exploration

BackgroundsIncreasing evidences have shown that reactive oxygen species(ROS) are very important mediators in liver ischemia/reperfusion injury(IRI). ROS include hydrogen peroxide (H₂O₂), superoxide anion (O-2) and hydroxyl radical (HO), which could be generated by activated Kupffer cells in the liver and contribute to reperfusion injury. The injury occurs in different two periods: The acute injury phase(the early phase), which is characterized by liver injury occurring within 6h after reperfusion the injury is associated with Kupffer cell activation, the release of the pro- inflammatory cytokines, and generation of reactive oxygen species. Ischemia activates Kupffer cells, which are the main sources of vascular reactive oxygen formation during the initial reperfusion period. The first phase is followed by the subsequent subacute-phase response (late phase), which is characterized by a massive neutrophil infiltration and further production of the inflammatory mediators. During the whole injury process, endothelial cells are the major target of the immunity injury. So the endothelium damage is the primary pathological change and willbe present in the early stage of the graft dysfunction. This study mainly focus on the injury of endothelial cells when challenged with reactive oxygen species or TNF, also figure out the mechanism of the process. This study will be value for the clinical therapy for graft dysfunction which began with endothelial cell injury.Inflammatory cytokines such as tumor necrosis factor(TNF) and reactive oxygen species (ROS) represent common mechanism of atherosclerosis. The primary cells limiting inflammation is vascular endothelial cell(s) (EC). The signal transduction and resulting gene expression in response to TNF/ROS in EC have been extensively studied. TNF and ROS (H₂O₂) in cultured cells activate overlapping of transcriptional factors and induce a common array of gene expression including EC adhesion molecules, coagulation factors, and matrix proteins leading to EC activation/dysfunction. However, TNF and ROS do have distinct effects on EC and induce distinct intracellular signaling. For example, TNF induced EC death/apoptosis only in the presence of protein synthesis inhibitor cycloheximide, which blocks TNF-induced NF-kB-dependent gene expression of survival factors. In contrast, H₂O₂ alone is sufficient to induce EC death. This is due, at least in part, to the fact that H₂O₂ induces the activation of c-Jun N-terminal kinase (JNK) but not of NF-kB in EC. Thus, it appears that stress-activated kinase cascade (JNK/p38) can be activated by both TNF and ROS. However, the signal transduction pathways utilized by H₂O₂ have not been fully defined.Apoptosis signal-regulating kinase 1 (ASK1), a member of the mitogen-activated protein kinase (MAPK) kinase kinase family, is an upstream activator of JNK and p38 MAPK signaling cascades. ASK1 can be activated in response to diverse stresses including proinflammatory cytokine TNF, ROS, and death receptor Fas, disruption of microtubule structures, protein aggregation in endoplasmic reticulum, and genotoxic stress from nucleus. In vitro data suggest that activation of ASK1 triggers various biological responses such as apoptosis, inflammation, differentiation, and survival in different cell types. Studies from ASK1-deficient mice indicate that ASK1 is critical for TNF and ROS-induced apoptosis signaling, suggesting that TNF andROS converge, at least, at the level of MAPK kinase kinase (ASK1). The mechanism by which TNF/ROS activates ASK1 is not fully understood. The identification of proteins associated with ASK1 has provided some insights. ASK1 is a 170-kDa protein that functionally is composed of an inhibitory N-terminal domain, an internal kinase domain, and a C-terminal regulatory domain. Several cellular factors including thioredoxin, glutaredoxin, and 14-3-3 have been reported to interacts with different ASK1 domains and inhibit ASK1 activity. TNF and H₂O₂ activate ASK1, in part, by dissociating these cellular inhibitors from ASK1. Several ASK1 upstream activators have been also identified. In response to TNF, the C-terminal domain of ASK1 binds to TNF receptor-associated factor 2 (TRAF2) and this association is required for ASK1 activation by TRAF2. It has been shown that ASK1 binding to TRAF2 may facilitate dissociation of ASK1 from thioredoxin. TRAF2 in turn induces ASK1 oligomerization, leading to autophosphorylation of ASK1 at Thr-845 within the activation loop in the kinase domain. Activated ASK1 subsequently recruits and activates its downstream targets MAP2K (MKK3/7 and MKK4/7) and MAPK (JNK and p38). Thus, ASK1 activation appears to involve several sequential steps including association of upstream activators (such as TRAF2 in response to TNF), release of cellular inhibitors (such as thioredoxin, glutaredoxin, glutathione S-transferase Mu, heat shock proteins, and 14-3-3), ASK1 oligomerization/autophosphorylation at Thr-845, and scaffold protein-mediated association of ASK1 with downstream MKK and JNK. However, the upstream activator mediating H₂O₂-induced ASK1 activation has not been identified.Protein kinase C (PKC) family has been implicated in the signal transduction pathways regulating a wide range of biological responses, including changes in cell morphology, differentiation, and proliferation . PKC family is divided into three groups, classic PKC(α,β, γ), novel PKC(μ, η, θ,δ,ε), and atypical PKC(ζ, λ),depending on their activation initiated by Ca2+, lipid second messengers, and/or protein activators. PKD1, a novel member of Ca2+/ calmodulin (CaM)-dependent kinase, differs from PKC by its overall structure, the catalytic domain sequence, and substrate specificity. The N-terminal part of PKD1 contains a pleckstrin homology (PH)domain and lacks the typical autoinhibitory pseudosubstrate motif present in PKCs. Recent studies suggest that PKD1 is important regulator of different intracellular signaling pathways.In this study, we employed pharmacological inhibitors and small interfering RNA (siRNA) approaches and identified PKD1 as a critical upstream mediator in H₂O₂ but not in TNF-induced ASK1 activation. Furthermore, we showed that 14-3-3 binding of 1 plays critical roles in mediating H2O2-induced ASK1-JNK activation and EC apoptosis.Although both tumor necrosis factor (TNF) and H₂O₂ induce activation of c-Jun N-terminal kinase (JNK) kinase cascades, it is not known whether they utilize distinct intracellular signaling pathways. In this study, we first examined a variety of pharmacological inhibitors on TNF and H₂O₂ induced JNK activation. GO6983 or staurosporine, which inhibits protein kinase C isoforms had no effects on TNF or H₂O₂ induced JNK activation. However, GO6976 and calphostin, which can inhibit protein kinase C as well as protein kinase D1 (PKD1), blocked H₂O₂ but not TNF-induced JNK activation, suggesting that PKD1 may be specifically involved in H₂O₂ induced JNK activation. Consistently, H₂O₂, but not TNF, induced phosphorylation of PKD1 and translocation of PKD1 from endothelial cell membrane to cytoplasm where it associates with the JNK upstream activator, apoptosis signal-regulating kinase 1 (ASK1). The association is mediated through the pleckstrin homology domain of PKD1 and the C-terminal domain of ASK1. Inhibition of PKD1 by GO6976 or by small interfering RNA of PKD1 blocked H₂O₂ induced ASK1-JNK activation and endothelial cell apoptosis. Interestingly, H₂O₂ induced 14-3-3 binding to PKD1 via the phospho-Ser-205/208 and phospho-Ser-219/223 and H₂O₂-induced 14-3-3 binding of PKD1 was specifically blocked by GO6976 but not by GO6983. More significantly, the 14-3-3-binding defective forms of PKD1 failed to associate with ASK1 and to activate JNK signaling, highlighting the importance of 14-3-3 binding of PKD1 in H₂O₂-induced activation of ASK1-JNK cascade. Thus, our data have identified PKD1 as a critical mediator in H₂O₂ but not TNF-induced ASK1-JNK signaling.Materials and methodsPart oneThe effect of PKD1 expression and activation on TNF or H₂O₂ induced ASK1-JNK signal transduction pathway1. BAEC culture (bovine aortic endothelial cell).2. A series of specific inhibitors of PKC or PKD1 pretreat BAEC cells, then stimulated with TNF or H₂O₂ respectively.3. Western blotting detect expression and activation of PKD1, ASK1, JNK. Phospho-specific antibody is used for the activation examination.4. Immunoflurescence detect the localization and translocation of PKD1 when challenged with TNF or H₂O₂.5. Different kinase activity PKD1 plasmid DNA transfected and fully expressed in cells, Western blotting examin downstream response.6. Down-regulate PKD1 expression with RNAi, Western blotting examin downstream response.Part twoPKD1 bind with ASK1 to form a complex through protein-protein interaction, the effect of this binding on ASK1-JNK pathway1. Transfect the construct to BAEC, fully expression.2. Immunoprecipitation check the complex between PKD1 and ASK1.3. Immunoflurescence detect the PKD1 and ASK1 co-localization.Part threeExplore the mechanism of PKD1 mediate H₂O₂ induced ASK1-JNK pathway1. GST Pull- down Assay: Prepare GST 14-3-3 fusion protein for detecting PKD1 bind with 14-3-3 protein.2. Site-directed mutagenesis: 14-3-3 binding sites S205/208 or S219/223 mutate to S205/208A or S219/223A.3. Immunoflurescence detect PKD1 localization and translocation in the cells under the different capacity of 14-3-3 binding.Part fourThe function of PKD1 mediate H₂O₂ induced ASK1-JNK pathway in endothelial cell1.ASK1 adenovirus preparation.2. Quantification of cell survival and apoptosis.Results Part onePKD1 mediate H₂O₂ induced ASK1-JNK pathway1. H₂O₂ can activate PKD1, but TNF can not.2. PKD1 specific inhibitor Go6976 and Calphostin can inhibit H₂O₂ induced ASK1-JNK activation, but can not inhibit TNF induced ASK1-JNK activation. Broad PKC inhibitors Go6983 and Staurosporin, which can not inhibit PKD1, neither can inhibit H₂O₂ nor TNF induced ASK1-JNK activation.3. PKDlSiRNA diminished PKD1 expression and down-regulate H₂O₂ induced ASK1-JNK activation.4. The PKD1 ΔPH is constitutive active form of PKD1, it can up- regulate H₂O₂ ASK1- JNK signal, whereas kinase dead PKD1 (PKD1KW) alleviate H₂O₂ induced ASK1- JNK downstream response.Part twoPKD1 and ASK1 form a complex and trigger downstream response1. PKD1 PH domain binds with C terminal of ASK1 and form a complex, this binding is PKD1 kinase dependent.2. 14-3-3 binding sites on PKD1 are mutated which block the binding between PKD1 and ASK1.3. PKD1 and ASK1 form a cytosolic complex in the cell, this is comfirmed by their co-localization.Part three14-3-3 is involved in the mechanism of PKD1 mediate H₂O₂ induced ASK1-JNK pathway1. 14-3-3 binds with PKD1 at the sites of PKD1 S205/208 and S219/223, this binding can be affected by PKD1 kinase activity.2. The different effections of inhibitors Go6976 and Go6983 on PKD1 primary activation sites S744/748 is inconsistant with their effections on ASK1-JNK activation.3. PKD1 specific inhibitors Go6976 can inhibit H₂O₂ induced 14-3-3 binding with PKD1, but broad PKC inhibitor Go6983 can not.4. PKD1S205/208A, PKD1S219/223A expression can directly enhance ASK1 14-3-3 binding site S967 phosphorylation, also diminish ASK1 downstream JNK response for H₂O₂.5. Like kinase dead PKD1, PKD1S205/208A, PKD1S219/223A which can not bind with 14-3-3, also did not translocate from membrane to cytosol when challenged with H₂O₂.Part fourPKD1 mediate H₂O₂ induced ASK1-JNK pathway paly a key role for endothelial cells apoptosis1. Inhibitor Go6976 can inhibit endothelial cells apoptosis, but Go6983 can not.2. PKD1 kinase activity is positively regulate endothelial cells apoptosis.3. PKDISiRNA decreased endothelium apoptosisConclusions1. PKD1 mediate H₂O₂ induced ASK1-JNK signal, the factors which can regulate PKD1 expression and activation also parallel affect ASK1 downstream response.2. PKD1 PH domain bind with C terminal of ASK1 and form a cytosolic complex, this binding is PKD1 kinase dependent and affected by 14-3-3 binding sites phosphorylation state.3. A model is present in PKD1 mediate H₂O₂ induced ASK1-JNK signal: PKD1 translocate from membrane to cytosol and form a complex with ASK1 when challenged with H₂O₂, after that, 14-3-3 is disassociate from ASK1 and move forward to PKD1, binding with PKD1, ASK1 is activated and pass the signal to downstream.4. The intervenor which decrease PKD1 expression or activity can definitely alleviate H₂O₂ induced endothelial cell apoptosis, whereas the methods enhance PKD1 activity can augment it.