Chi-Ming Liang

Class I and III phosphatidylinositol 3'-kinase play distinct roles in TLR signaling pathway.

PI3K involvement has been implicated in the TLR signal pathway. However, the precise roles of the different classes of PI3K in the pathway remain elusive. In this study, we have explored the functions of class I and class III PI3K in the TLR signal pathway using specific kinase mutants and PI3K lipid products. Our results reveal that class III PI3K specifically regulates CpG oligodeoxynucleotide (ODN)-induced cytokine and NO production as well as NF-κB activation, whereas class I PI3K regulates both CpG ODN- and LPS-induced IL-12 production and NF-κB activation. Additional studies of CpG ODN uptake with flow cytometric analysis show that class III PI3K, but not class I, regulates cellular CpG ODN uptake. Furthermore, experiments with MyD88-overexpressing fibroblast cells transfected with dominant-negative mutants of PI3K demonstrate that class III PI3K regulates CpG ODN-mediated signaling upstream of MyD88, while class I PI3K regulation is downstream of MyD88. These results suggest that class I and class III PI3K play distinct roles in not only the uptake of CpG ODN, but also responses elicited by CpG ODN and LPS.

Study of Capsular Polysaccharide from Vibrio parahaemolyticus

ABSTRACT The leading cause of food poisoning in both Taiwan and Japan is Vibrioparahaemolyticus infection, whose mechanism of enteropathogenesis is still unclear. To evaluate whether surface components are responsible for the intestinal adhesion of V. parahaemolyticus, we have developed a novel method for isolating the capsular polysaccharide (CPS) from V. parahaemolyticus (serotype O4:K8). We found that culturing of V. parahaemolyticus in broth for 1 week or more changed the colony form of the bacteria on an agar plate from opaque to translucent. The translucent colonies of V. parahaemolyticus contained little CPS and exhibited a much lower level of adherence to epithelial cells (Int-407) than the opaque colonies of the bacteria. Incubation of V. parahaemolyticus in medium supplemented with bile increased the levels of CPS and adherence. Treatment of V. parahaemolyticus with anti-CPS but not anti-LPS serum decreased the level of bacterial adherence. In addition, purified CPS bound to epithelial cells in a dose-dependent manner. Intranasal administration of CPS to mice in the presence of adjuvants such as immunostimulatory sequence oligodeoxynucleotides or cholera toxin elicited CPS-specific mucosal and systemic immune responses. These results indicate that CPS plays an important role in the adherence of V. parahaemolyticus to its target cells and may be considered a potential target for the development of a vaccine against this pathogen.

CpG-B oligodeoxynucleotide promotes cell survival via up-regulation of Hsp70 to increase Bcl-xL and to decrease apoptosis-inducing factor translocation.

Unmethylated CpG oligodeoxynucleotides (ODNs) activate immune responses in a TLR9-dependent manner. In this study, stimulation of mouse macrophages with CpG-B ODN increased cellular Hsp70 expression and prevented apoptosis induced by serum starvation or staurosporine treatment. CpG-B ODN-induced Hsp70 expression depended on TLR9, MyD88, and phosphatidylinositol 3-kinase. Inhibition of Hsp70 synthesis by an inhibitor (quercetin) or antisense hsp70 attenuated not only Hsp70 expression but also the anti-apoptotic capacity of CpG-B ODN. Ectopic expression of Hsp70 rescued the inhibitory effect of quercetin on CpG-B ODN-induced anti-apoptosis. Additional experiments demonstrated that quercetin and anti-sense hsp70 modulated CpG-B ODN-induced anti-apoptosis via a caspase-3-independent pathway by down-regulating the survival gene bcl-xL and by increasing translocation of apoptosis-inducing factor. These findings suggest that CpG-B ODN may up-regulate Hsp70 via a TLR9/MyD88/phosphatidylinositol 3-kinase pathway to increase Bcl-xL and to decrease apoptosis-inducing factor nuclear translocation, resulting in anti-apoptosis.

MIG-7 Controls COX-2/PGE2-Mediated Lung Cancer Metastasis

More effective treatments for metastatic lung cancer remain a pressing clinical need. In this study, we identified migration inducting gene-7 (MIG-7) protein as critical for COX-2/prostaglandin E2 (PGE2)- and Akt/GSK-3β-dependent tumor invasion/metastasis. COX-2/PGE2 activated EP4 to enhance Akt and GSK-3β phosphorylation and β-catenin/T-cell factor/lymphoid enhancer factor signaling leading to MIG-7 upregulation. RNAi-mediated attenuation of MIG-7 blocked COX-2/PGE2- and Akt/GSK-3β-mediated migration/invasion effects. Furthermore, MIG-7 protein inhibited protein phosphatase 2A to sustain Akt/GSK-3β phosphorylation and cancer-cell migration/invasion. Cancer cells overexpressing MIG-7 exhibited increased expression of ZEB-1 and Twist in parallel with epithelial-mesenchymal transition, metastasis and cancer lethality. MIG-7 protein level positively correlated with advanced stages of human lung cancers. MIG-7 thus offers a theranostic target for cancer metastases arising from aberrant activation of the cellular COX-2/PGE2 and Akt/GSK-3β signaling pathways.

Enhancement of the immunity to foot-and-mouth disease virus by DNA priming and protein boosting immunization.

Subunit vaccination is effective in eliciting humoral responses to a variety of viral antigens, however, it has not generated persistent protective immunity to foot-and-mouth disease virus (FMDV). In this study, we observed that priming mice with a DNA plasmid encoding VP1 of the FMDV O/Taiwan/97 capsid protein followed by boosting with a VP1 peptide conjugate (P29-KLH) resulted in production of not only high titers of antibodies but also antibodies with FMDV neutralizing activities. Moreover, the mice immunized in this manner cleared the virus from their sera in FMDV challenge experiments. Mice subjected to DNA plasmid priming and P29-KLH protein boosting had relatively higher ratio of IgG2a/IgG1 than those primed and boosted with P29-KLH conjugate. Addition of an oligodeoxynucleotide (ODN) containing immunostimulatory cytosine-phosphate-guanosine (CpG) motifs to P29-KLH conjugate also induced a higher ratio of IgG2a/IgG1 and significantly higher titer of neutralizing antibodies. These results indicate that treating animals with DNA plasmids priming and FMDV antigen(s) boosting may elicit immunity to FMD and this immune response may be augmented by CpG ODN.

Induction of immunity in swine by purified recombinant VP1 of foot-and-mouth disease virus.

VP1, a capsid protein of foot-and-mouth disease virus (FMDV), contains neutralizing epitopes of the virus. Due to its poor water solubility, recombinant Escherichia coli derived VP1 (rVP1) has previously been used mainly in a denatured form and is not well characterized. Here, using SDS to assist protein refolding and then removing SDS with a detergent removing column, we have successfully purified rVP1 in two aqueous-soluble forms, i.e. monomer and dimer. Studies showed that dimerization occurs by an inter-molecular disulfide bond between two cysteine residues at position 187 of each monomer. Heat treatment revealed that rVP1 dimer exhibited a more thermal-stable conformation than the monomeric form. Both monomeric and dimeric rVP1 reacted with anti-FMDV antibodies. Immunization studies demonstrated that vaccination of swine with either forms of rVP1 was effective in generating immune responses and protecting them from viral challenge.

Involvement of Heat Shock Protein (Hsp)90β but Not Hsp90α in Antiapoptotic Effect of CpG-B Oligodeoxynucleotide

Unmethylated CpG oligodeoxynucleotides (CpG ODNs) activate immune responses in a TLR9-dependent manner. In this study, we found that stimulation of mouse macrophages and dendritic cells with B-type CpG ODN (CpG-B ODN) increased the cellular level of heat shock protein (Hsp) 90β but not Hsp90α and prevented apoptosis induced by serum starvation or staurosporine treatment. The CpG-B ODN-induced Hsp90β expression depended on TLR9, MyD88, and PI3K. Inhibition of Hsp90β level by expressing small-interfering RNA suppressed not only Hsp90β expression but also PI3K-dependent phosphorylation of Akt and CpG-B ODN-mediated antiapoptosis. Additional studies demonstrated that as described by other group in mast cells, Hsp90β but not Hsp90α was associated with Bcl-2. Inhibition of Hsp90β suppressed the CpG-B ODN-induced association of Hsp90β with Bcl-2 and impaired the inhibitory effect of CpG-B ODN in the release of cytochrome c and activation of caspase-3. This study thus reveals the involvement of Hsp90β but not Hsp90α in CpG-B ODN-mediated antiapoptotic response and that Hsp90β is distinct from Hsp90α in regulation of the cellular function of immune cells.

DNA vaccination against foot-and-mouth disease via electroporation: study of molecular approaches for enhancing VP1 antigenicity.

Foot‐and‐mouth disease virus (FMDV) affects susceptible livestock animals and causes disastrous economic impact. Immunization with plasmid expressing VP1 that contains the major antigenic epitope(s) of FMDV as cytoplasmic protein (cVP1) failed to elicit full protection against FMDV challenge.

F-spondin plays a critical role in murine neuroblastoma survival by maintaining IL-6 expression.

F‐spondin is associated with the regulation of axonal growth and the development of the nervous system. Its mechanism of action, however, is not clearly understood. In this study, we found that murine neuroblastoma Neuro‐2a cells expressed a significant level of IL‐6, but only trace amounts of IL‐12, tumor necrosis factor α and nitric oxide. Knock‐down of F‐spondin mRNA in murine neuroblastoma NB41A3 and Neuro‐2a cells using small interfering RNAs led to decreased IL‐6 levels along with lower resistance to serum starvation and cytotoxic amyloid β1–42 (Aβ1–42) peptide. Restoring decline of F‐spondin or IL‐6 induced by F‐spondin knock‐down through adding exogenous F‐spondin, IL‐6 or over‐expressing F‐spondin reversed the cell death induced by Aβ1–42 peptide or serum starvation. The decrease of IL‐6 level was positively correlated with decrease of NF‐κB and inhibition of p38 mitogen‐activated protein kinase (MAPK). Over‐expressing MEKK, a kinase activator of the p38 MAPK pathway, increased IL‐6 production, restored the decrease of p38 induced by F‐spondin knock‐down, and rescued the cells from death caused by Aβ1–42 peptide. Taken together, these results suggest that F‐spondin may play a critical role in murine neuroblastoma survival under adverse conditions by maintaining IL‐6 level via a MEKK/p38 MAPK/NF‐κB‐dependent pathway.

Albumin fibrillization induces apoptosis via integrin/FAK/Akt pathway

BackgroundNumerous proteins can be converted to amyloid-like fibrils to increase cytotoxicity and induce apoptosis, but the methods generally require a high concentration of protein, vigorous shaking, or fibril seed. As well, the detailed mechanism of the cytotoxic effects is not well characterized. In this study, we have developed a novel process to convert native proteins into the fibrillar form. We used globular bovine serum albumin (BSA) as a model protein to verify the properties of the fibrillar protein, investigated its cellular effects and studied the signaling cascade induced by the fibrillar protein.ResultsWe induced BSA, a non-cytotoxic globular protein, to become fibril by a novel process involving Superdex-200 column chromatography in the presence of anionic or zwittergenic detergent(s). The column pore size was more important than column matrix composite in fibril formation. The fibrillar BSA induced apoptosis in BHK-21 cell as well as breast cancer cell line T47D. Pre-treating cells with anti-integrin antibodies blocked the apoptotic effect. Fibrillar BSA, but not globular BSA, bound to integrin, dephosphorylated focal adhesion kinase (FAK), Akt and glycogen synthase kinase-3β (GSK-3β).ConclusionWe report on a novel process for converting globular proteins into fibrillar form to cause apoptosis by modulating the integrin/FAK/Akt/GSK-3β/caspase-3 signaling pathway. Our findings may be useful for understanding the pathogenesis of amyloid-like fibrils and applicable for the development of better therapeutic agents that target the underlying mechanism(s) of the etiologic agents.