Chang-Kwon Lee

Identification of the most active interleukin-32 isoform.

Cytokines are crucial in host defence against pathogens such as bacteria, viruses, fungi and parasites. A newly described cytokine, interleukin‐32 (IL‐32), induces various proinflammatory cytokines (tumour necrosis factor‐α, IL‐1β, IL‐6) and chemokines in both human and mouse cells through the nuclear factor‐κB and p38 mitogen‐activated protein kinase inflammatory signal pathway. The IL‐32 primarily acts on monocytic cells rather than T cells. In an attempt to isolate the IL‐32 soluble receptor, we used an IL‐32 ligand‐affinity column to purify neutrophil proteinase 3, which is a serine proteinase involved in many inflammatory diseases. IL‐32 has biological activity associated with Mycobacterium tuberculosis and chronic proinflammatory diseases such as rheumatoid arthritis. IL‐32 is transcribed as six alternative splice variants and the biological activity of each individual isoform remains unknown. Here, we cloned the complementary DNA of the four IL‐32 isoforms (α, β, γ and δ) that are the most representative IL‐32 transcripts. To produce recombinant protein with a high yield, the amino acids of two cysteine residues were mutated to serine residues, because serine residues are not conserved among different species. The multi‐step purified recombinant IL‐32 isoform proteins were assessed for their biological activities with different cytokine assays. The γ isoform of IL‐32 was the most active, although all isoforms were biologically active. The present study will provide a specific target to neutralize endogenous IL‐32, which may contribute to basic and clinical immunology.

Gene Transfer of Redox Factor-1 Inhibits Neointimal Formation: Involvement of Platelet-Derived Growth Factor-β Receptor Signaling via the Inhibition of the Reactive Oxygen Species–Mediated Syk Pathway

The role of apurinic/apyrimidinic endonuclease-1/redox factor-1 (Ref-1) in vascular smooth muscle cells has yet to be clearly elucidated. Therefore, we attempted to determine the roles of Ref-1 in the migration induced by platelet-derived growth factor (PDGF)-BB and in its signaling in rat aortic smooth muscle cells (RASMCs). Cellular migration, superoxide (O2−·) production, Rac-1 activity, and neointima formation were determined in cells transfected with adenoviruses encoding for Ref-1 (AdRef-1) and small interference RNA of Ref-1. Overexpression of Ref-1 induced by treatment with RASMCs coupled with AdRef-1 inhibited the migration induced by PDGF-BB. PDGF-BB also increased the phosphorylation of the PDGF&bgr; receptor, spleen tyrosine kinase (Syk), mitogen-activated protein kinase, and heat shock protein 27, but these increases were significantly inhibited by AdRef-1 treatment. PDGF-BB increased O2−· production and Rac-1 activity, and these were diminished in cells transfected with AdRef-1. In contrast, RASMC migration, phosphorylation of Syk and O2−· production in response to PDGF-BB were increased by the knock down of Ref-1 with small interference RNA. The phosphorylation of PDGF&bgr; receptor in response to PDGF-BB was inhibited completely by the Syk inhibitor and was partly attenuated by a NADPH oxidase inhibitor. PDGF-BB increased the sprout outgrowth of the aortic ring ex vivo, which was inhibited in the AdRef-1–infected RASMCs as compared with the controls. Balloon injury–induced neointimal formation was significantly attenuated by the gene transfer of AdRef-1. These results indicate that Ref-1 inhibits the PDGF-mediated migration signal via the inhibition of reactive oxygen species–mediated Syk activity in RASMCs.

Diminished expression of dihydropteridine reductase is a potent biomarker for hypertensive vessels

To identify the new targets for hypertension, we analyzed the protein expression profiles of aortic smooth muscle in spontaneously hypertensive rats (SHR) of various ages during the development of hypertension, as well as in age‐matched normotensive Wistar–Kyoto (WKY) rats, using a proteomic analysis. The expressions of seven proteins were altered in SHR compared with WKY rats. Of these proteins, NADH dehydrogenase 1α, GSTω1, peroxi‐redoxin I and transgelin were upregulated in SHR compared with WKY rats. On the other hand, the expression of HSP27 and Ran protein decreased in SHR. The diminution of dihydrobiopterin reductase, an enzyme located in the regeneration pathways of tetrahydrobiopterin (BH4), was also prominent in SHR. The results from a PCR analysis revealed that the expression of BH4 biosynthesis enzymes – GTP cyclohydrolase‐1 and sepiapterin reductase – decreased and increased, respectively, in SHR compared with WKY rats. The level of BH4 was less in aortic strips from SHR than from WKY rats. Moreover, treatment with BH4 inhibited aortic smooth muscle contraction induced by serotonin. These results suggest that the deficiency in BH4 regeneration produced by diminished dihydrobiopterin reductase expression is involved in vascular disorders in hypertensive rats.

Identification of the varR Gene as a Transcriptional Regulator of Virginiamycin S Resistance in Streptomyces virginiae

A gene designated varR (for virginiae antibiotic resistance regulator) was identified in Streptomyces virginiae 89 bp downstream of a varS gene encoding a virginiamycin S (VS)-specific transporter. The deduced varR product showed high homology to repressors of the TetR family with a conserved helix-turn-helix DNA binding motif. Purified recombinant VarR protein was present as a dimer in vitro and showed clear DNA binding activity toward the varS promoter region. This binding was abolished by the presence of VS, suggesting that VarR regulates transcription of varS in a VS-dependent manner. Northern blot analysis revealed that varR was cotranscribed with upstream varS as a 2.4-kb transcript and that VS acted as an inducer of bicistronic transcription. Deletion analysis of the varS promoter region clarified two adjacent VarR binding sites in the varS promoter.

γ-Butyrolactone autoregulators and receptor proteins in non-Streptomyces actinomycetes producing commercially important secondary metabolites

Abstract. The presence of γ-butyrolactone autoregulators and their receptor proteins were investigated in five representative strains of non-Streptomyces actinomycetes producing commercially important secondary metabolites. Ethyl acetate extracts of culture were assayed using wild-type S. virginiae for virginiae butanolide, S. lavendulae FRI-5 for IM-2, and S. griseus HH1 for A-factor. Actinoplanes teichomyceticus and Amycolatopsis mediterranei were shown to produce autoregulators. Corresponding autoregulator-binding activities were found in the crude cell-free lysates of these strains, using the binding assay with tritium-labeled autoregulator analogues as ligands, which suggests that non-Streptomyces actinomycetes might have autoregulator-dependent signaling cascades.

p38 MAPK Participates in Muscle-Specific RING Finger 1-Mediated Atrophy in Cast-Immobilized Rat Gastrocnemius Muscle

Skeletal muscle atrophy is a common phenomenon during the prolonged muscle disuse caused by cast immobilization, extended aging states, bed rest, space flight, or other factors. However, the cellular mechanisms of the atrophic process are poorly understood. In this study, we investigated the involvement of mitogen-activated protein kinase (MAPK) in the expression of muscle-specific RING finger 1 (MuRF1) during atrophy of the rat gastrocnemius muscle. Histological analysis revealed that cast immobilization induced the atrophy of the gastrocnemius muscle, with diminution of muscle weight and cross-sectional area after 14 days. Cast immobilization significantly elevated the expression of MuRF1 and the phosphorylation of p38 MAPK. The starvation of L6 rat skeletal myoblasts under serum-free conditions induced the phosphorylation of p38 MAPK and the characteristics typical of cast-immobilized gastrocnemius muscle. The expression of MuRF1 was also elevated in serum-starved L6 myoblasts, but was significantly attenuated by SB203580, an inhibitor of p38 MAPK. Changes in the sizes of L6 myoblasts in response to starvation were also reversed by their transfection with MuRF1 small interfering RNA or treatment with SB203580. From these results, we suggest that the expression of MuRF1 in cast-immobilized atrophy is regulated by p38 MAPK in rat gastrocnemius muscles.

Cloning and Functional Analysis by Gene Disruption of a Gene Encoding a γ-Butyrolactone Autoregulator Receptor from Kitasatospora setae

Gamma-butyrolactone autoregulator receptors of the genus Streptomyces have a common activity as DNA-binding transcriptional repressors, controlling secondary metabolism and/or morphological differentiation. A gene encoding a gamma-butyrolactone autoregulator receptor was cloned from a bafilomycin B1 producer, Kitasatospora setae, for the first time from a non-Streptomyces genus of actinomycetes, and its function was evaluated by in vitro and in vivo analyses. The gene fragment was initially cloned by PCR with primers designed from two highly conserved regions of Streptomyces autoregulator receptors (BarA, FarA, ScbR, and ArpA), followed by genomic Southern hybridization yielding a 7-kb BamHI fragment on which a 654-bp receptor gene (ksbA) was identified. The recombinant KsbA protein demonstrated clear binding activity toward 3H-labeled autoregulators, especially toward [3H]SCB1, confirming that ksbA encodes a real autoregulator receptor of K. setae. To clarify the in vivo function of ksbA, a ksbA-disrupted strain was constructed by means of homologous recombination after introducing a ksbA disruption construct via transconjugation from Escherichia coli. No difference in morphology was found between the wild-type strain and the ksbA disruptants. However, the ksbA disruptants started producing bafilomycin 18 h earlier than the wild-type strain and showed a 2.4-fold-higher accumulation of bafilomycin. The phenotype was restored to the original wild-type phenotype by complementation with intact ksbA, indicating that the autoregulator receptor protein of K. setae acts as a primary negative regulator of the biosynthesis of bafilomycin but plays no role in cytodifferentiation of K. setae. This indicates that, unlike the A-factor receptor of Streptomyces griseus, the autoregulator receptor (ksbA) of K. setae belongs to a family of autoregulator receptors which control secondary metabolism but play no role in morphological differentiation.

DJ-1/park7 protects against neointimal formation via the inhibition of vascular smooth muscle cell growth

AIMS DJ-1/park7 is a ubiquitously expressed multifunctional protein that plays essential roles in a variety of cells. However, its function in the vascular system has not been determined. We investigated the protective roles of DJ-1/park7 in vascular disorders, especially in neointimal hyperplasia. METHODS AND RESULTS DJ-1/park7 was strongly expressed in the neointimal layer, in which its oxidized form was predominant. Treatment of vascular smooth muscle cells (VSMCs) from the mouse aorta with H(2)O(2) increased the oxidation of DJ-1/park7 visualized on two-dimensional electrophoresis gels. The growth of VSMCs in FBS-containing media and the release of H(2)O(2) were significantly increased in DJ-1/park7(-/-) knockout mice compared with DJ-1/park7(+/+) wild-type mice. The expression of cyclin D1 and the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 were greater in VSMCs from the DJ-1/park7(-/-) aorta than from the DJ-1/park7(+/+) aorta. Both of these measures were inhibited by treatment with an ERK1/2 inhibitor or antioxidants and in DJ-1/park7-overexpressing cells. VSMC proliferation, cyclin D1 expression, and ERK1/2 phosphorylation in response to platelet-derived growth factor-BB were upregulated in DJ-1/park7(-/-) compared with DJ-1/park7(+/+) mice. VSMCs of DJ-1/park7(-/-) mice exhibited higher levels of sprout outgrowth of aortic strips and neointimal plaque formation elicited by carotid artery ligation compared with those of DJ-1/park7(+/+) mice. CONCLUSION These results indicate that DJ-1/park7 is involved in the growth of VSMCs, thereby inhibiting neointimal hyperplasia, and suggest that it might play protective roles in vascular remodelling.

3-Morpholinosydnonimine participates in the attenuation of neointima formation via inhibition of annexin A2-mediated vascular smooth muscle cell migration

3‐Morpholinosydnonimine (SIN‐1) affects vascular smooth muscle cell migration and proliferation, processes essential for atherosclerosis. However, the mechanism by which SIN‐1 exerts these effects has not been elucidated. We used 2‐DE followed by MALDI‐TOF/TOF MS to identify responses in protein expression to SIN‐1 in rat aortic smooth muscle. Platelet‐derived growth factor‐BB increased cell migration and proliferation in rat aortic smooth muscle cells, and subsequent SIN‐1 treatment inhibited it. Administration of SIN‐1 in vivo attenuated neointima formation in balloon‐injured rat carotid arteries. Proteomic analysis showed that glutathione peroxidase and 40S ribosomal protein S12 were differentially expressed in aortic strips exposed to SIN‐1. Expression of annexin A2 was decreased by SIN‐1. Platelet‐derived growth factor‐BB‐induced cell migration was increased and inhibited in rat aortic smooth muscle cells with overexpression and knockdown of annexin A2 gene, respectively. The expression of annexin A2 was increased in vascular neointima compared with the intact control, which was inhibited by SIN‐1 treatment. These results demonstrate that SIN‐1 may attenuate vascular neointima formation by inhibiting annexin A2‐mediated migration. Therefore, annexin A2 may be a potential target for therapeutic strategies for atherosclerosis.

Does phosphorylation of cofilin affect the progression of human bladder cancer

BackgroundWe determined the differently expressed protein profiles and their functions in bladder cancer tissues with the aim of identifying possible target proteins and underlying molecular mechanisms for taking part in their progression.MethodsWe examined the expression of proteins by proteomic analysis and western blot in normal urothelium, non-muscle-invasive bladder cancers (NMIBCs), and muscle-invasive bladder cancers (MIBCs). The function of cofilin was analyzed using T24 human bladder cancer cells.ResultsThe expression levels of 12 proteins were altered between bladder cancers and normal bladder tissues. Of these proteins, 14-3-3σ was upregulated in both NMIBCs and MIBCs compared with controls. On the other hand, myosin regulatory light chain 2, galectin-1, lipid-binding AI, annexin V, transthyretin, CARD-inhibitor of NF-κB-activating ligand, and actin prepeptide were downregulated in cancer samples. Cofilin, an actin-depolymerizing factor, was prominent in both NMIBCs and MIBCs compared with normal bladder tissues. Furthermore, we confirmed that cofilin phosphorylation was more prominent in MIBCs than in NMIBCs using immunoblotting and immunohistochemcal analyses. Epidermal growth factor (EGF) increased the phosphorylation of cofilin and elevated the migration in T24 cells. Knockdown of cofilin expression with small interfering RNA attenuated the T24 cell migration in response to EGF.ConclusionsThese results demonstrate that the increased expression and phosphorylation of cofilin might play a role in the occurrence and invasiveness of bladder cancer. We suspected that changes in cofilin expression may participate in the progression of the bladder cancer.