Kangkai Wang

The inhibition of LPS-induced production of inflammatory cytokines by HSP70 involves inactivation of the NF-κB pathway but not the mapk pathways

ABSTRACT The objective of this study was to evaluate the negative regulatory role of heat shock protein 70 (HSP70) on endotoxin-induced activation of inflammatory cytokine signaling pathways in a macrophage cell line. Our studies show that elevation of HSP70 either by activation of the heat shock response (HSR) or through forced expression of the hsp70.1 gene downregulates cytokine expression. Our experiments showed that activation of the HSR and HSP70 overexpression could inhibit LPS-mediated expression of the proinflammatory cytokines TNF-&agr; and IL-1 at the mRNA and protein levels. We also investigated the effects of HSP70 elevation on signaling pathways downstream of LPS and its receptors, including the NF-&kgr;B and mitogen-activated protein kinase (MAPK) pathways. The effects of HSP70 on cytokine expression were correlated with its effects on activation of NF-&kgr;B, a known activator of the tnf&agr; and Il-1 genes. Overexpression of HSP70 inhibited the nuclear translocation of p65, the transcriptionally active component of the NF-&kgr;B complex, and prevented the degradation of I&kgr;B&agr;, the regulator of NF-&kgr;B activity. However, HSP70 elevation did not markedly inhibit signaling through the MAPK arm of the LPS-induced pathway, suggesting that the effects of HSP70 are mediated primarily through the NF-&kgr;B cascade. Our experiments therefore suggested that elevated levels of HSP70 inhibit LPS-induced production of inflammatory cytokines by a mechanisms involving inactivation of NF-&kgr;B but cast doubt on significant role for the MAPK pathway in these effects.

Nuclear heat shock protein 72 as a negative regulator of oxidative stress (hydrogen peroxide)-induced HMGB1 cytoplasmic translocation and release.

In response to inflammatory stimuli (e.g., endotoxin, proinflammatory cytokines) or oxidative stress, macrophages actively release a ubiquitous nuclear protein, high-mobility group box 1 (HMGB1), to sustain an inflammatory response to infection or injury. In this study, we demonstrated mild heat shock (e.g., 42.5°C, 1 h), or enhanced expression of heat shock protein (Hsp) 72 (by gene transfection) similarly rendered macrophages resistant to oxidative stress-induced HMGB1 cytoplasmic translocation and release. In response to oxidative stress, cytoplasmic Hsp72 translocated to the nucleus, where it interacted with nuclear proteins including HMGB1. Genetic deletion of the nuclear localization sequence (NLS) or the peptide binding domain (PBD) from Hsp72 abolished oxidative stress-induced nuclear translocation of Hsp72-ΔNLS (but not Hsp72-ΔPBD), and prevented oxidative stress-induced Hsp72-ΔPBD-HMGB1 interaction in the nucleus. Furthermore, impairment of Hsp72-ΔNLS nuclear translocation, or Hsp72-ΔPBD-HMGB1 interaction in the nucleus, abrogated Hsp72-mediated suppression of HMGB1 cytoplasmic translocation and release. Taken together, these experimental data support a novel role for nuclear Hsp72 as a negative regulator of oxidative stress-induced HMGB1 cytoplasmic translocation and release.

Heat shock response inhibits release of high mobility group box 1 protein induced by endotoxin in murine macrophages.

The purpose of this study was to evaluate the kinetic changes and the localization of high-mobility group box 1 protein (HMGB1) and to observe the effect of heat shock response (HSR) on the expression and release of HMGB1 in lipopolysaccharide (LPS)-activated murine macrophage-like RAW 264.7 cells. Reverse transcriptase (RT)-PCR and Western blot were used to examine HMGB1 expression after LPS treatment. The intracellular localization of HMGB1 in normal or LPS-activated cells was investigated by immunocytochemical analysis and HMGB1 released from cultured macrophages by Western blot. HSR was performed by incubating RAW 264.7 cells at 42.5°C for 1 h then recovery at 37°C for 12 h. The effect of HSR on expression and release of HMGB1 was observed. The results showed that a decrease of HMGB1 mRNA expression was observed at 18 h after LPS (500 ng/mL) treatment, although the total intracellular HMGB1 protein levels were not affected. A visible translocation of HMGB1 from the nuclear to the cytoplasm was observed at 20 h after stimulation with LPS (500 ng/mL). Furthermore, HMGB1 was released into the medium by LPS-activated RAW 264.7 cells in a time- and dose-dependent manner. Heat shock pretreatment significantly inhibited LPS-induced release of HMGB1 and the translocation of HMGB1 from the nucleus to the cytoplasm in RAW 264.7 cells. These findings suggest that the release of HMGB1 by LPS-activated macrophages is a late event in the pathogenesis of sepsis and that HSR could inhibit the release and translocation of HMGB1 induced by LPS.

ATP‐binding domain of heat shock protein 70 is essential for its effects on the inhibition of the release of the second mitochondria‐derived activator of caspase and apoptosis in C2C12 cells

Hydrogen peroxide (H2O2) is a well known oxidative stress inducer causing apoptosis of many cells. Previously, we have shown that heat shock pretreatment blocked the release of the second mitochondria‐derived activator of caspase (Smac) to the cytosol and inhibited apoptosis of C2C12 myoblast cells in response to H2O2. The present study aimed to elucidate the underlying mechanism by over‐expressing a major stress‐inducible protein, heat shock protein (HSP) 70, and characterizing the resulting cellular changes. We demonstrate that HSP70 over‐expression markedly inhibited the release of Smac and prevented the activation of caspases‐9 and ‐3 and apoptosis in C2C12 cells under H2O2 treatment. However, no direct interaction between HSP70 and Smac was observed by co‐immunoprecipitation. Mutational analysis demonstrated that the ATP‐binding domain of HSP70, rather than the peptide‐binding domain, was essential for these observed HSP functions. Taken together, our results provide evidence supporting the role of HSP70 in the protection of C2C12 cells from H2O2‐induced and Smac‐promoted apoptosis by preventing the release of Smac from mitochondria, thereby inhibiting activation of caspases‐9 and ‐3. This mechanism of HSP70 action is dependent on its ATP‐binding domain but independent of its interaction with Smac protein.

Nucleolin involved in myocardial ischaemic preconditioning via post-transcriptional control of HSPA1A expression

AIMSnRecent studies have identified the critical roles of nucleolin in a variety of cellular processes, including regulation of viral replication and tumour formation. However, the possible roles of nucleolin in myocardial preconditioning remain undefined.nnnMETHODS AND RESULTSnWe used an in vivo rat myocardial ischaemic preconditioning (IP) model (four cycles of 5 min ischaemia and 10 min reperfusion) and cellular hydrogen peroxide preconditioning (H2O2-PC) models. We found that nucleolin mRNA and protein expression showed a time-dependent increase during the recovery of myocardial ischaemic preconditioning in rats and H2O2-PC in neonatal rat cardiomyocytes. Nucleolin overexpression enhanced the protective effects of H2O2-PC, whereas nucleolin ablation abrogated the H2O2-PC-mediated protection in cardiomyocytes. On the other hand, nucleolin overexpression increased the stabilization of the HSPA1A mRNA and the expression of HSPA1A protein in cardiomyocytes, whereas nucleolin ablation abrogated the up-regulation of HSPA1A induced by H2O2-PC in cardiomyocytes. An interaction between nucleolin and HSPA1A mRNA was further identified using the RNA-protein interaction studies. Reporter gene assays, which depended on the untranslated regions (UTR) of HSPA1A mRNA, revealed that the post-transcriptional regulation was mainly attributed to the 3 UTR. Finally, HSPA1A anti-sense oligonucleotides (asODNs) attenuated the protective effect of nucleolin in cardiomyocytes.nnnCONCLUSIONnThese results indicate that nucleolin is up-regulated and involved in myocardial protection of ischaemic preconditioning via a post-transcriptional control of HSPA1A expression.

ANGIOGENESIS INHIBITOR ENDOSTATIN PROTECTS MICE WITH SEPSIS FROM MULTIPLE ORGAN DYSFUNCTION SYNDROME.

ABSTRACT Endostatin is an endogenous inhibitor of vascular endothelium. It can inhibit endothelial cell migration, proliferation, and vascular angiogenesis and is mainly used for anticancer therapy. We have previously found that endostatin is an important node protein in the pathogenesis of sepsis. However, its impacts on sepsis have not yet been reported. We established a septic mouse model using cecal ligation and puncture (CLP) and gave the mice either endostatin or placebo (saline). The effects of endostatin on serum enzyme, Evans blue leakage, lung wet-to-dry weight ratio, and cytokine (tumor necrosis factor &agr;, interleukin 1&bgr; [IL-1&bgr;], and IL-6) production were assessed. Survival rates were observed for up to 3 days. In addition, we examined the effects of endostatin on serum vascular endothelial growth factor A (VEGF-A), VEGF-C, and pathological changes and scores of lung tissues as well as the phosphorylation of JNK, p38, and ERKl/2 proteins in lung tissues of mice with sepsis. We found that endostatin can increase the survival of septic mice in a time- and dose-dependent manner probably by reducing multiorgan dysfunctions shown by serum indicators, morphologic changes, Evans blue leakage, wet-to-dry weight ratio, and inflammation of lung tissues. In addition, endostatin could reduce serum tumor necrosis factor &agr;, IL-1&bgr;, IL-6, and VEGF-C levels in septic mice as well as inhibit phosphorylation of p38 and ERK1/2 in lung tissues of septic mice. This is the first study demonstrating the protective effect of endostatin on sepsis and its possible underlying mechanisms from the aspects of inhibiting inflammatory responses, blocking VEGF receptor, attenuating VEGF-C expression, and reducing vascular permeability. Overall, the study revealed the potential protect role for endostatin in the treatment of sepsis.

Identification of potential biomarkers by serum proteomics analysis in rats with sepsis.

ABSTRACT This study was aimed to find new biomarkers for diagnosis and prediction of prognosis of sepsis. Serum samples from nonsurvivor, survivor, and control groups were obtained at 12 h after the induction of sepsis and labeled with isobaric tags (iTRAQ) and then analyzed by two-dimensional liquid chromatography and tandem mass spectrometry. Protein identification and quantification were obtained using mass spectrometry and the ProteinPilot software. Bioinformatics annotation was performed by searching against the PANTHER database. Enzyme-linked immunosorbent assays were used to further confirm the protein identification and differential expression. A logistic regression was then used to screen the index set for diagnosis and prognosis of sepsis. We found that 47 proteins were preferentially elevated in septic rats (both nonsurvivors and survivors) compared with the control rats, and 28 proteins were preferentially elevated in the NS rats as compared with the S group. Several biomarkers, such as multimerin 1, ficolin 1, carboxypeptidase N (CPN2), serine protease 1, and platelet factor 4, were tightly correlated with the diagnosis of sepsis. Logistic regression analyses established multimerin 1, pro–platelet basic protein, fibrinogen-&agr;, and fibrinogen-&bgr; for prognosis of sepsis.

Interleukin-1 Receptor 2: A New Biomarker for Sepsis Diagnosis and Gram-negative/gram-positive Bacterial Differentiation.

ABSTRACT This study was undertaken to explore the value of interleukin-1 receptor 2 (IL1R2) as a novel potential biomarker for diagnosis of sepsis and discrimination of gram-negative (G−)/gram-positive (G+) bacterial sepsis. The study was performed in Kunming mice and septic patients. Inactive Escherichia coli or Staphylococcus aureus were used to stimulate Kunming mice (109u200aCFU/kg). In clinical study, septic patients with different pathogen infection were studied, and healthy volunteers and patients with systemic inflammatory response syndrome without definite infection were enrolled as control. IL1R2 transcriptions of human subjects’ peripheral leukocytes were measured by real-time quantitative polymerase chain reaction assay. IL1R2 serum concentrations of mice and human subjects were measured by enzyme-linked immunosorbent assay. The value of IL1R2 as a biomarker was compared with procalcitonin (PCT), C-reactive protein (CRP), and Acute Physiology and Chronic Health Evaluation II (APACHE II). The results showed that IL1R2 expression was upregulated in mice treated with inactive Escherichia coli and septic patients. The elevation of serum IL1R2 was more significant in septic patients infected by Escherichia coli or G− bacteria than in those infected by Staphylococcus aureus or G+ bacteria. For sepsis diagnosis and G−/G+ bacterial sepsis discrimination, serum IL1R2 was more sensitive and specific than the traditional biomarkers such as PCT, CRP, and APACHE II as shown by the receiver operating characteristic curves. It was suggested that IL1R2 was a potential biomarker for diagnosis and G−/G+ bacterial differentiation in sepsis.

Intracellular Delivery of Recombinant alpha B-crystallin into Neonatal Rat Cardiomyocytes has a Protective Effect on the Cells

In order to deliver alpha B-crystallin (alpha B-C) into cardiomyocytes and study its cellular protection, the full-length cDNA fragment encoding human alpha B-C was cloned into the bacterial expression vector pGEX-MTS containing the base sequence of membrane-translocating sequence (MTS) which mediates intracellular delivery of peptides and expressed as a fusion protein coupled to glutathione S-transferase (GST).After glutathione affinity chromatography and cleaved from GST by factor Xa, the recombinant MTS- alpha B-C was separated from GST and factor Xa by anion exchange chromatography. Recombinant MTS- alpha B-C was characterized by SDS-PAGE and Western immunoblot analysis. The purified MTS- alpha B-C migrated on SDS-PAGE as a single band to an apparent molecular weight (Mr.23kD) that corresponded to total native alpha B-C plus MTS, and was recognized on Western immunoblot by anti-human alpha B-crystallin antibody. MTS- alpha B-C displayed chaperone-like function in an ATP-containing buffer at 37℃ by disaggregating the denatured and aggregated actin induced by hydrogen peroxide (H2O2 )treatment. It was observed under fluorescence microscope that FITC-labeled MTS- alpha B-C had gone into neonatal rat cardiomyocytes by MTS mediation after the cells were incubated with it for 6 hours while FITC-labeled alpha B-C and bovine serum albumin had not gone into the cells. Recombinant MTS- alpha B-C is not cytotoxic, and MTS- alpha B-C-treated cells displayed increased H2O2-tolerance compared with non-treated cells.