Gang Min Hur

Caffeic acid phenethyl ester inhibits nitric oxide synthase gene expression and enzyme activity

Since nitric oxide (NO) synthesized by inducible nitric oxide synthase (iNOS) has been known to be involved in inflammatory and autoimmune-mediated tissue destruction, modulation of NO synthesis or action represents a new approach to the treatment of inflammatory and autoimmune diseases. Caffeic acid phenethyl ester (CAPE), an active component of honeybee propolis, has been identified to show anti-inflammatory, anti-viral and anti-cancer activities. The present study, therefore, examined effects of CAPE on iNOS expression and activity of iNOS enzyme itself. Treatment of RAW 264.7 cells with CAPE significantly inhibited NO production and iNOS protein expression induced by lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma). CAPE also inhibited iNOS mRNA expression and nuclear factor-kappa B (NF-kappaB) binding activity in a concentration-dependent manner. Furthermore, transfection of RAW 264.7 cells with iNOS promoter linked to a chloramphenicol acetyltransferase reporter gene, revealed that CAPE inhibited the iNOS promoter activity induced by LPS plus IFN-gamma through the NF-kappaB sites of the iNOS promoter. In addition, CAPE directly interfered with the catalytic activity of murine recombinant iNOS enzyme. These results suggest that CAPE may exert its anti-inflammatory effect by inhibiting the iNOS gene expression at the transcriptional level through the suppression of NF-kappaB activation, and by directly inhibiting the catalytic activity of iNOS.

The orphan nuclear receptor SHP acts as a negative regulator in inflammatory signaling triggered by Toll-like receptors

The orphan nuclear receptor SHP (small heterodimer partner) is a transcriptional corepressor that regulates hepatic metabolic pathways. Here we identified a role for SHP as an intrinsic negative regulator of Toll-like receptor (TLR)-triggered inflammatory responses. SHP-deficient mice were more susceptible to endotoxin-induced sepsis. SHP had dual regulatory functions in a canonical transcription factor NF-κB signaling pathway, acting as both a repressor of transactivation of the NF-κB subunit p65 and an inhibitor of polyubiquitination of the adaptor TRAF6. SHP-mediated inhibition of signaling via the TLR was mimicked by macrophage-stimulating protein (MSP), a strong inducer of SHP expression, via an AMP-activated protein kinase–dependent signaling pathway. Our data identify a previously unrecognized role for SHP in the regulation of TLR signaling.

Regulation of 3-Phosphoinositide-dependent Protein Kinase-1 (PDK1) by Src Involves Tyrosine Phosphorylation of PDK1 and Src Homology 2 Domain Binding

3-Phosphoinositide-dependent protein kinase-1 (PDK1) appears to play a central regulatory role in many cell signalings between phosphoinositide-3 kinase and various intracellular serine/threonine kinases. In resting cells, PDK1 is known to be constitutively active and is further activated by tyrosine phosphorylation (Tyr9 and Tyr373/376) following the treatment of the cell with insulin or pervanadate. However, little is known about the mechanisms for this additional activation of PDK1. Here, we report that the SH2 domain of Src, Crk, and GAP recognized tyrosine-phosphorylated PDK1 in vitro. Destabilization of PDK1 induced by geldanamycin (a Hsp90 inhibitor) was partially blocked in HEK 293 cells expressing PDK1-Y9F. Co-expression of Hsp90 enhanced PDK1-Src complex formation and led to further increased PDK1 activity toward PKB and SGK. Immunohistochemical analysis with anti-phospho-Tyr9 antibodies showed that the level of Tyr9 phosphorylation was markedly increased in tumor samples compared with normal. Taken together, these data suggest that phosphorylation of PDK1 on Tyr9, distinct from Tyr373/376, is important for PDK1/Src complex formation, leading to PDK1 activation. Furthermore, Tyr9 phosphorylation is critical for the stabilization of both PDK1 and the PDK1/Src complex via Hsp90-mediated protection of PDK1 degradation.

Midazolam inhibits proinflammatory mediators in the lipopolysaccharide-activated macrophage.

Background:Midazolam, a benzodiazepine, has a hypnotic effect and is widely used as a sedative. The role of midazolam in activation of macrophages during sepsis is not known. The aim of this study was to evaluate the antiinflammatory actions of midazolam in cultured macrophages. Methods:Using a macrophage cell line, RAW264.7 cells, the effect of midazolam on proinflammatory mediators and activation of mitogen-activated protein kinase was measured by Western blot. Nuclear factor-&kgr;B (NF-&kgr;B) activation and translocation of p65 subunit of NF-&kgr;B was measured using luciferase assay and immunocytochemistry. Superoxide production was measured by lucigenin chemiluminescence. Results:Midazolam significantly inhibited lipopolysaccharide-induced up-regulation of both cyclooxygenase 2 and inducible nitric oxide synthase in a dose-dependent manner (approximately 3–30 &mgr;m). I&kgr;B-α degradation and NF-&kgr;B transcriptional activity induced by lipopolysaccharide were also suppressed by the midazolam. Nuclear translocation of the p65 subunit of NF-&kgr;B was inhibited by midazolam. Furthermore, midazolam suppressed phosphorylation of p38 mitogen-activated protein kinase and also inhibited lipopolysaccharide-induced superoxide production in macrophages. Conclusions:These results suggest that midazolam has an antiinflammatory action by inhibiting inducible nitric oxide synthase and cyclooxygenase-2 expression, possibly through suppression of NF-&kgr;B and p38 mitogen-activated protein kinase activation.

Association of LETM1 and MRPL36 Contributes to the Regulation of Mitochondrial ATP Production and Necrotic Cell Death

Leucine zipper/EF hand-containing transmembrane-1 (LETM1) is a mitochondrial inner membrane protein that was first identified in Wolf-Hirschhorn syndrome, and was deleted in nearly all patients with the syndrome. LETM1 encodes for the human homologue of yeast Mdm38p, which is a mitochondria-shaping protein of unclear function. Here, we describe LETM1-mediated regulation of mitochondrial ATP production and biogenesis. We show that LETM1 overexpression can induce necrotic cell death in HeLa cells, in which LETM1 reduces mitochondrial biogenesis and ATP production. LETM1 acts as an anchor protein and associates with mitochondrial ribosome protein L36. Adenovirus-mediated overexpression of LETM1 reduced mitochondrial mass and expression of many mitochondrial proteins. LETM1-mediated inhibition of mitochondrial biogenesis enhanced glycolytic ATP supply and activated protein kinase B activity and cell survival signaling. The expression levels of LETM1 were significantly increased in multiple human cancer tissues compared with normals. These data suggest that LETM1 serves as an anchor protein for complex formation with the mitochondrial ribosome and regulates mitochondrial biogenesis. The increased expression of LETM1 in human cancer suggests that dysregulation of LETM1 is a key feature of tumorigenesis.

Ethanol extract of propolis inhibits nitric oxide synthase gene expression and enzyme activity.

Propolis obtained from honeybee hives has been used in Oriental folk medicine as an anti-inflammatory, anti-carcinogenic, or immunomodulatory agent. However, the molecular basis for anti-inflammatory properties of propolis has not yet been established. Since nitric oxide (NO) synthesized by inducible nitric oxide synthase (iNOS) has been known to be involved in inflammatory and autoimmune-mediated tissue destruction, modulation of NO synthesis or action represents a new approach to the treatment of inflammatory and autoimmune diseases. The present study, therefore, examined effects of ethanol extract of propolis (EEP) on iNOS expression and activity of iNOS enzyme itself. Treatment of RAW 264.7 cells with EEP significantly inhibited NO production and iNOS protein expression induced by lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma). EEP also inhibited iNOS mRNA expression and nuclear factor-kappa B (NF-kappaB) binding activity in a concentration-dependent manner. Furthermore, transfection of RAW 264.7 cells with iNOS promoter linked to a chloramphenicol acetyltransferase (CAT) reporter gene, revealed that EEP inhibited the iNOS promoter activity induced by LPS plus IFN-gamma through the NF-kappaB sites of the iNOS promoter. In addition, EEP directly interfered with the catalytic activity of murine recombinant iNOS enzyme. These results suggest that EEP may exert its anti-inflammatory effect by inhibiting the iNOS gene expression via action on the NF-kappaB sites in the iNOS promoter and by directly inhibiting the catalytic activity of iNOS.

NOVEL PRE-C/C GENE MUTANTS OF HEPATITIS B VIRUS IN CHRONIC ACTIVE HEPATITIS : NATURALLY OCCURRING ESCAPE MUTANTS

We have analysed serum samples taken from hepatitis B virus (HBV) e antigen (HBeAg)-positive and HBeAg-negative chronic active hepatitis (CAH) patients by PCR using primers spanning the pre-core/core (C) and pre-S1/S2 ORFs. Nucleotide sequence analysis showed that among 18 HBV-infected CAH patients, 11 had virus with a G to A mutation (nucleotide 1896; leading to the formation of a stop codon) and one patient also had virus with an additional G to A mutation three nucleotides downstream (nucleotide 1899). HBV from three patients that were HBeAg-negative showed a 1 bp deletion at nucleotide 1937, causing pre-termination of the C gene. Mutation frequencies in the sequences identified as coding for cytotoxic T lymphocyte epitopes, B cell epitopes, CD4+ helper T cell epitopes and arginine-rich regions of the HBV C peptide were investigated. Mutations were more frequently identified in these regions, suggesting that the mutations might have been selected as a result of immune responses.

PHF20 regulates NF-κB signalling by disrupting recruitment of PP2A to p65

Constitutive NF-κB activation in cancer cells is caused by defects in the signalling network responsible for terminating the NF-κB response. Here we report that plant homeodomain finger protein 20 (PHF20) maintains NF-κB in an active state in the nucleus by inhibiting the interaction between PP2A and p65. We show that PHF20 induces canonical NF-κB signalling by increasing the DNA-binding activity of NF-κB subunit p65. In PHF20 overexpressing cells, the termination of tumour necrosis factor-induced p65 phosphorylation is impaired whereas upstream signalling events triggered by tumour necrosis factor are unaffected. This effect strictly depends on the interaction between PHF20 and methylated lysine residues of p65, which hinders recruitment of PP2A to p65, thereby maintaining p65 in a phosphorylated state. We further show that PHF20 levels correlate with p65 phosphorylation levels in human glioma specimens. Our work identifies PHF20 as a novel regulator of NF-κB activation and suggests that elevated expression of PHF20 may drive constitutive NF-κB activation in some cancers.

Modulatory role of phospholipase D in the activation of signal transducer and activator of transcription (STAT)-3 by thyroid oncogenic kinase RET/PTC

BackgroundRET/PTC (rearranged in transformation/papillary thyroid carcinomas) gene rearrangements are the most frequent genetic alterations identified in papillary thyroid carcinoma. Although it has been established that RET/PTC kinase plays a crucial role in intracellular signaling pathways that regulate cellular transformation, growth, and proliferation in thyroid epithelial cells, the upstream signaling that leads to the activation of RET/PTC is largely unknown. Based on the observation of high levels of PLD expression in human papillary thyroid cancer tissues, we investigated whether PLD plays a role in the regulating the RET/PTC-induced STAT3 activation.MethodsCancer tissue samples were obtained from papillary thyroid cancer patients (n = 6). The expression level of PLD was examined using immunohistochemistry and western blotting. Direct interaction between RET/PTC and PLD was analyzed by co-immunoprecipitation assay. PLD activity was assessed by measuring the formation of [3H]phosphatidylbutanol, the product of PLD-mediated transphosphatidylation, in the presence of n-butanol. The transcriptional activity of STAT3 was assessed by m67 luciferase reporter assay.ResultsIn human papillary thyroid cancer, the expression levels of PLD2 protein were higher than those in the corresponding paired normal tissues. PLD and RET/PTC could be co-immunoprecipitated from cells where each protein was over-expressed. In addition, the activation of PLD by pervanadate triggered phosphorylation of tyrosine 705 residue on STAT-3, and its phosphorylation was dramatically higher in TPC-1 cells (from papillary carcinoma) that have an endogenous RET/PTC1 than in ARO cells (from anaplastic carcinoma) without alteration of total STAT-3 expression. Moreover, the RET/PTC-mediated transcriptional activation of STAT-3 was synergistically increased by over-expression of PLD, whereas the PLD activity as a lipid hydrolyzing enzyme was not affected by RET/PTC.ConclusionThese findings led us to suggest that the PLD synergistically functions to activate the STAT3 signaling by interacting directly with the thyroid oncogenic kinase RET/PTC.

Prevention of TNF-induced necrotic cell death by rottlerin through a Nox1 NADPH oxidase.

Previous studies have demonstrated that rottlerin, a specific PKCδ inhibitor, potentiates death receptormediated apoptosis through a cytochrome c-dependent or -independent pathway. However, its ability to regulate necrotic cell death, as well as the underlying mechanism, remains unknown. We found that in murine fibrosarcoma L929 cells, treatment with rottlerin protected the cells against TNF-induced necrosis, whereas it sensitized the cells to apoptosis induced by co-treatment with Hsp90 inhibitor geldanamycin and TNF, in a manner independent of its ability to inhibit PKC-δ. TNF treatment induced rapid accumulation of mitochondrial superoxide (O2-) through the Nox1 NADPH oxidase when cells undergo necrosis. Moreover, pretreatment with rottlerin failed to induce the GTP-bound form of small GTPase Rac1 by TNF treatment, and subsequently suppressed mitochondrial O2- production and poly(ADP-ribose) polymerase activation, thus inhibiting necrotic cell death. Therefore, our study suggests that Nox1 NADPH oxidase is a new molecular target for anti-necrotic activity of rottlerin upon death-receptor ligation.