Berberine Suppresses Fibronectin Expression through Inhibition of c-Jun Phosphorylation in Breast Cancer Cells / 한국유방암학회지

PURPOSE: The exact mechanism regulating fibronectin (FN) expression in breast cancer cells has not been fully elucidated. In this study, we investigated the pharmacological mechanism of berberine (BBR) with respect to FN expression in triple-negative breast cancer (TNBC) cells. METHODS: The clinical significance of FN mRNA expression was analyzed using the Kaplan-Meier plotter database (http://kmplot.com/breast). FN mRNA and protein expression levels were analyzed by real-time polymerase chain reaction and western blotting, respectively. RESULTS: Using publicly available clinical data, we observed that high FN expression was associated with poor prognosis in patients with breast cancer. FN mRNA and protein expression was increased in TNBC cells compared with non-TNBC cells. As expected, recombinant human FN significantly induced cell spreading and adhesion in MDA-MB231 TNBC cells. We also investigated the regulatory mechanism underlying FN expression. Basal levels of FN mRNA and protein expression were downregulated by a specific activator protein-1 (AP-1) inhibitor, SR11302. Interestingly, FN expression in TNBC cells was dose-dependently decreased by BBR treatment. The level of c-Jun phosphorylation was also decreased by BBR treatment. CONCLUSION: Our findings demonstrate that FN expression is regulated via an AP-1–dependent mechanism, and that BBR suppresses FN expression in TNBC cells through inhibition of AP-1 activity.

Troglitazone Inhibits Matrix Metalloproteinase-9 Expression and Invasion of Breast Cancer Cell through a Peroxisome Proliferator-Activated Receptor γ-Dependent Mechanism / 한국유방암학회지

PURPOSE: Peroxisome proliferator-activated receptor γ (PPARγ) is involved in the pathology of numerous diseases including atherosclerosis, diabetes, obesity, and cancer. Matrix metalloproteinases (MMPs) play a significant role in tissue remodeling related to various processes such as morphogenesis, angiogenesis, tissue repair, invasion, and metastasis. We investigated the effects of PPARγ on MMP expression and invasion in breast cancer cells. METHODS: MCF-7 cells were cultured and then cell viability was monitored in an MTT assay. Western blotting, gelatin zymography, real-time polymerase chain reaction, and luciferase assays were performed to investigate the effect of the synthetic PPARγ ligand troglitazone on MMP expression. Transcription factor DNA binding was analyzed by electrophoretic mobility shift assay. A Matrigel invasion assay was used to assess the effects of troglitazone on MCF-7 cells. RESULTS: Troglitazone did not affect MCF-7 cell viability. 12-O-tetradecanoylphorbol-13-acetate (TPA) induced MMP-9 expression and invasion in MCF-7 cell. However, these effects were decreased by troglitazone. TPA increased nuclear factor κB and activator protein-1 DNA binding, while troglitazone inhibited these effects. The selective PPARγ antagonist GW9662 reversed MMP-9 inhibition by troglitazone in TPA-treated MCF-7 cells. CONCLUSION: Troglitazone inhibited nuclear factor κB and activator protein-1-mediated MMP-9 expression and invasion of MCF-7 cells through a PPARγ-dependent mechanism.

The Effect of Adiponectin on the Regulation of Filaggrin Expression in Normal Human Epidermal Keratinocytes

BACKGROUND: Adiponectin, an adipokine secreted from adipocytes, affects energy metabolism and also shows anti-diabetic and anti-inflammatory properties. Recent studies have reported that adiponectin plays a role in regulating skin inflammation. OBJECTIVE: This study aimed to investigate the effect of adiponectin on the expression of filaggrin (FLG) in normal human epidermal keratinocytes (NHEKs). METHODS: NHEKs were serum-starved for 6h before being treated with adiponectin. Afterward, cell viability was assessed by MTT assay. We also treated with calcium, interleukin (IL)-4, and IL-13 to provide positive and negative comparative controls, respectively. Gene mRNA expression was quantified using real time reverse transcription polymerase chain reaction, and protein expression was evaluated using Western blot. To evaluate the relationship among mitogen-activated protein kinases (MAPKs), activator protein 1 (AP-1), and FLG, we also treated cells with inhibitors for MAPKs JNK, p38, and ERK1/2. RESULTS: FLG and FLG-2 mRNA expression in NHEKs significantly increased after treatment with 10 µg/ml adiponectin. Adiponectin also restored FLG and FLG-2 mRNA expression that was otherwise inhibited by treatment with IL-4 and IL-13. Adiponectin induced FLG expression via AP-1 and MAPK signaling. CONCLUSION: Adiponectin positively regulated the expression of FLG and could be useful as a therapeutic agent to control diseases related to disrupted skin barrier function.

Optimization of culture medium for scarless healing of fetal mouse wounds and analysis of regions with active cell proliferation / 中华皮肤科杂志

Objective To optimize the culture medium for scarless wound healing in outbred fetal Kunming (KM) mice,and to locate and preliminarily analyze the region with active cell proliferation.Methods After 6 pregnant mice were sacrificed at the gestational age of 15 days,a total of 180 skin grafts were obtained from the back of 60 fetal mice,and wounds on the skin grafts were made uniformly with minimally invasive sterile device.Then,these skin grafts with wounds were randomly and equally divided into 6 groups to be treated with high-glucose Dulbecco's modified Eagle's medium (DMEM),DMEM + 5％ fetal bovine serum (FBS),DMEM + 10％ FBS,low-glucose Eagle's minimum essential medium (MEM),MEM + 5％ FBS,and MEM + 10％ FBS for 3 days,respectively.Then,these skin grafts were embedded in optimal cutting temperature (OCT) compound,and subjected to hematoxylin and eosin (HE) staining to select the optimal culture condition for wound healing.Under the optimal culture condition,the region with active cell proliferation was located by labeling and tracking cutaneous stem cells with 5-ethynyl-2-deoxyuridine (EdU),and activator protein-1 (AP-1) expression was detected by immunofluorescent staining.Results After 3-day cultivation,wound healing rates significantly differed among the DMEM group,DMEM + 5％ FBS group,DMEM + 10％ FBS group,MEM group,MEM + 5％ FBS group and MEM + 10％ FBS group (0,3.33％,6.67％,3.33％,46.67％ and 26.67％,respectively,x2 =41.39,P ＜ 0.05).Additionally,the MEM + 5％ FBS group showed a significantly higher wound healing rate than the other groups except the MEM + 10％ FBS group (all P ＜ 0.01).Logistic regression analysis showed that the type of basal medium (MEM:OR =11.717,95％ CI:3.274-41.934,P ＜ 0.001) and FBS concentrations (5％ FBS:OR =24.625,95％ CI:3.027-200.299,P =0.003;10％ FBS:OR =13.449,95％ CI:1.618-111.813,P =0.016) were factors influencing fetal wound healing.Under the culture condition of 5％ FBS + MEM,the dermis and epidermis healed well without epidermal thickening.The cutaneous stem cell labeling technique showed that the papillary dermis and basal layer of the epidermis were the two major regions with active cell proliferation in the wound of fetal mouse skin.Moreover,AP-1 was expressed abundantly in these two regions as well.Conclusions The culture condition of 5％ FBS + MEM is considered to be optimal for in vitro wound healing in fetal mice.The papillary dermis and basal layer of the epidermis are two major regions with active cell proliferation during wound healing in fetal mouse skin,and play important roles in wound healing process.

Crotonis Fructus Extract Inhibits 12-O-Tetradecanoylphorbol-13-Acetate-Induced Expression of Matrix Metalloproteinase-9 via the Activator Protein-1 Pathway in MCF-7 Cells / 한국유방암학회지

PURPOSE: Metastatic cancers spread from the primary site of origin to other parts of the body. Matrix metalloproteinase-9 (MMP-9) is essential in metastatic cancers owing to its major role in cancer cell invasion. Crotonis fructus (CF), the mature fruits of Croton tiglium L., have been used for the treatment of gastrointestinal disturbance in Asia. In this study, the effect of the ethanol extract of CF (CFE) on MMP-9 activity and the invasion of 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated MCF-7 cells was examined. METHODS: The cell viability was evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The expression of MMP-9 was examined by Western blotting, zymography, and real-time polymerase chain reaction. An electrophoretic mobility gel shift assay was performed to detect activator protein-1 (AP-1) DNA binding activity and cell invasiveness was measured by an in vitro Matrigel invasion assay. RESULTS: CFE significantly suppressed MMP-9 expression and activation in a dose-dependent manner. Furthermore, CFE attenuated the TPA-induced activation of AP-1. CONCLUSION: The results indicated that the inhibitory effects of CFE against TPA-induced MMP-9 expression and MCF-7 cell invasion were dependent on the protein kinase C δ/p38/c-Jun N-terminal kinase/AP-1 pathway. Therefore, CFE could restrict breast cancer invasiveness owing to its ability to inhibit MMP-9 activity.

Effects of penehyclidine hydrochloride on activities of NF-κB and AP-1 during actue lung injury induced by blunt chest trauma-hemorrhagic shock and resuscitation in rats / 中华麻醉学杂志

Objective To investigate the effects of penehyclidine hydrochloride on activities of nuclear factor kappa B (NF-kB) and activator protein-1 (AP-1) during actue lung injury induced by blunt chest trauma-hemorrhagic shock and resuscitation (HSR) in rats.Methods Thirty male Sprague-Dawley rats,aged 8 weeks,weighing 250-300 g,were randomly assigned into 3 equal groups (n =10 each) using a random number table:sham operation group (group S),blunt chest trauma-HSR group (group THSR) and penehyclidine hydrochloride group (group PHCD).The model of actue lung injury induced by blunt chest trauma-HSR was induced by dropping a 300 g weight onto a precordium in anesthetized rats.Blood was withdrawn via the femoral artery 5 min later until MAP was decreased to 35-45 mmHg within 15 min and maintained at this level for 60 min,followed by resuscitation.In PHCD group,PHCD 2 mg/kg was injected intravenously at 60 min after hemorrhagic shock.At 6 h after the model was established,blood samples were obtained for measurement of concentrations of tumor necrosis factor-alpha (TNF-α) in serum.The lungs were then removed for determination of lung water content,myeloperoxidase (MPO) activaty (by colorimetric assay),NF-κB and AP-1 activaties (using electrophoretic mobility shift assay) in lung tissues,and for microscopic examination of pathologic changes (under light microscope).The left lung was lavaged,and lung permeability index (LPI) was calculated.Results Compared with S group,lung water content,LPI,serum TNF-α level and activites of MPO,NF-κB and AP-1 were significantly increased in THSR and PHCD groups.Compared with THSR group,lung water content,LPI,serum TNF-α concentrations and activites of MPO,NF-κB and AP-1 were significantly decreased in PHCD group.The pathological damage to lung tissues was significantly reduced in PHCD group as compared with THSR group.Conclusion PHCD can inhibit activities of NF-κB and AP-1 in lung tissues,thus mitigating acute lung injury induced by blunt chest trauma-HSR in rats.

Roles of osteopontin and nuclear transcription factor in chronic cyclosporine nephrotoxicity / 第二军医大学学报

Objective: To investigate the roles of osteopontin (OPN) and nuclear transcription factor in a rat model of chronic cyclosporine A (CsA) nephrotoxicity. Methods: Male Sprague-Dawley rats maintained on a low salt diet were treated daily with vehicle (olive oil, 1 mL • kg-1 • d-1, sc.) and CsA (15 mL • kg-1 • d-1, sc.) for 4 weeks. Renal histopathology was estimated by trichrome staining (tubulointerstitial fibrosis) and immunohistochemistry (ED-1) to assess the degrees of renal tubulointerstitial lesions. In addition, OPN mRNA and protein expression and nuclear transcription factor (NF-κB and AP-1) were studied by northern blotting analysis, immunohistochemistry, electrophoretic mobility shift assay, and immunoblotting analysis. Results: CsA-treated rats displayed significantly striped tubulointerstitial fibrosis ([38. 9 ± 3. 3]%/5 mm2 vs [0± 0]%/5 mm2, P<0. 01) and increased ED-1-positive cells (89±9 vs 7±2, P<0. 01). Compared with VH-treated rats, CsA-treated rats showed significantly upregulated OPN mRNA and protein expression in the proximal tubular cells, mainly localizing at areas of severe injured tissues. CsA-treated group also had significantly increased activities of NF-κB ([218±19]% vs [ 116± 15] %, P<0. 01) and AP-1 ([735±225]% vs[101±4]%, P<0. 01), and significantly decreased expression of ([9 ± 7]% vs [105±7]%, P<0. 01). Correlation analysis revealed that upregulated OPN mRNA was positively correlated with tubulointerstitial fibrosis (r= 0.959, P<0. 001) and activities of NF-κB and AP-1 (NF-κB: r = 0.773, P<0. 01; AP-1: r =0.619, P = 0. 01, respectively). Conclusion: Our findings suggest that OPN, nuclear transcription factor NF-κB and AP-1 are involved in renal tubulointerstitial injury in chronic CsA nephrotoxicity.

Role of activator protein-1 in electro-acupuncture-induced up-regulation of heme oxygenase-1 expression in lung tissues in a rabbit model of endotoxic shock / 中华麻醉学杂志

Objective To evaluate the role of activator protein-1 (AP-1 ) in electro-acupuncture (EA )-induced up-regulation of heme oxygenase-1 (HO-1 ) expression in lung tissues in a rabbit model of endotoxic shock .Methods Seventy healthy male New Zealand white rabbits ,aged 2 months ,weighing 1.5-2.0 kg ,were randomly divided into 7 groups ( n=10 each ) using a random number table :normal control group (group C ) , endotoxic shock-induced acute lung injury (ALI ) group (group ALI ) , EA + ALI group (group EL ) , non-acupoint+EA+ ALI group (group NEL ) , curcumin (HO-1 inhibitor ) group (group Cur ) , dimethyl sulfoxide group (group D ) ,and EA+ALI+curcumin group (group ELC ) .Bilateral 15 min EA stimulation of Zusanli and Feishu (according to atlas of animals acupoints ) was performed (frequency 15Hz ) once a day for 5 consecutive days before lipopolysaccharide (LPS ) administration in EL and ELC groups ,while in group NEL ,EA stimulation was performed at non-acupoints located 0.5 cm lateral to Zusanli and Feishu acupoints with the same parameters . The animals were anesthetized with urethane and tracheostomized .The animals kept spontaneous breathing .Right internal carotid artery was cannulated for blood pressure monitoring . Ear vein was cannulated for drug administration .At 5 days after EA stimulation ,curcumin 25 mg/kg (in 0.5 ml of 0.1% dimethyl sulfoxide ) was injected in Cur and ELC groups ,0.1% dimethyl sulfoxide 0.5 ml was injected in D group ,and the equal volume of normal saline was given in the other groups .LPS 5 mg/kg (in 2 ml of 0.9% normal saline ) was injected intravenously at 30 min after administration in ALI ,EL ,NEL and ELC groups ,while the equal volume of normal saline was given in the other three groups .Endotoxic shock was confirmed by decrease in mean arterial pressure to 75% of the baseline value within 2 h after LPS injection .Blood samples were collected from the common carotid artery at 6 h after LPS or normal saline administration and the rabbits were then sacrificed .The lungs were removed for microscopic examination and the pathological changes were scored .The wet/dry lung weight ratio (W/D ratio ) was calculated .The malondialdehyde (MDA ) content and superoxide dismutase (SOD ) activity in lung tissues were detected ,and the expression of HO-1 mRNA ,HO-1 ,c-Jun mRNA and c-Jun in lung tissues was determined by Western blot .Results Compared with group C ,the pathological score ,W/D ratio and MDA content were significantly increased ,and the SOD activity was decreased in ALI ,EL ,NEL and ELC groups ,the expression of HO-1 mRNA ,HO-1 ,c-Jun mRNA and c-Jun was up-regulated in groups ALI ,EL and NEL ( P0.05) .There was no significant difference in the parameters mentioned above between Cur and D groups ( P>0.05 ) .Compared with group ALI ,the pathological score ,W/D ratio and MDA content were significantly decreased ,and SOD activity was increased ,and the expression of HO-1 mRNA and HO-1 was up-regulated in EL and ELC groups ( P0.05) .The pathological score ,W/D ratio and MDA content were significantly higher ,and the SOD activity and expression of HO-1 mRNA ,HO-1 ,c-Jun mRNA and c-Jun were lower in group ELC than in group EL ( P<0.05) .Conclusion EA up-regulates HO-1 expression through activating AP-1 during endotoxic shock-induced ALI in rabbits ,thus protecting the lung .

Thioredoxin inhibits human vascular endothelial cell adhesion molecules expression via Smad3/AP-1 pathway / 中华老年医学杂志

Objective To investigate the molecular mechanisms of protective effects of thioredoxin (Trx) on human vascular endothelial cells in atherosclerosis.Methods The cell models of Trx-overexpressing cells (Ad Trx) and the control cells (Ad-con) were established by adenovirus vector gene transfer technology in human umbilical vein endothelial cells (HUVECs).The oxidized low density lipoprotein,a risk factor of atherosclerosis,was used as a stimulator.Western blot and indirect immunofluorescence were used to detect the protein expression levels and the cellular localization of Trx,adhesion molecules (ICAM-1,VCAM-1) and the upstream signal pathways.Trx activity was detected by insulin disulfide reduction assay,and cellular reactive oxygen species (ROS)production was detected by fluorescent probe DCFH-DA.Results As compared with control group,Trx protein expression level was enhanced in Ad-trx group and the Trx activity in Ad-Trx group was upregulated by (26.2 ±3.3)％.The result of ROS detection showed that overexpression of Trx significantly inhibited the cellular ROS generation.As compared with control group,overexpression of Trx obviously inhibited the adhesion molecules expression but markedly promoted the phosphorylation of Smad3 in endothelial cells with or without oxLDL stimulation (P＜0.05).Pretreatment of cells with SIS3,a specific inhibitor of Smad3 phosphorylation,reversed Trx-induced inhibition of adhesion molecules expression.Further studies showed that pretreatment of cells with SIS3 enhanced oxLDL-induced AP-1 subunit c-fos nuclear expression.Conclusions The enhancement of Smad3 phosphorylation and c-Fos nuclear expression are mainly responsible for the Trx-induced downregulation of adhesion molecules.

Galectin-3 increases the motility of mouse melanoma cells by regulating matrix metalloproteinase-1 expression

Although mounting evidence indicates the involvement of galectin-3 in cancer progression and metastasis, the underlying molecular mechanisms remain largely unknown. In this study, we investigated the effect and possible mechanism of galectin-3 on the migration and invasion of B16F10, a metastatic melanoma cell line, in which galectin-3 and matrix metalloproteinase-1 (MMP-1) were both found to be highly expressed. Knockdown of galectin-3 with specific siRNA reduced migration and invasion, which was associated with reduced expression of MMP-1. To further investigate the underlying mechanism, we examined the effect of galectin-3 knockdown on the activity of AP-1, a transcriptional factor regulating MMP-1 expression. We found that galectin-3 directly interacted with AP-1 and facilitated the binding of this complex to the MMP-1 promoter that drives MMP-1 transcription. Moreover, silencing of galectin-3 inhibited binding of fra-1 and c-Jun to promoter sites of MMP-1 gene. Consistent with these in vitro findings, our in vivo study demonstrated that galectin-3 shRNA treatment significantly reduced the total number of mouse lung metastatic nodules. Taken together, galectin-3 facilitates cell migration and invasion in melanoma in vitro and can induce metastasis in vivo, in part through, regulating the transcription activity of AP-1 and thereby up-regulating MMP-1 expression.

Inhibitory effect of (-)-epigallocatechin gallate on titanium particle-induced TNF-alpha release and in vivo osteolysis

Tumor necrosis factor-alpha (TNF-alpha) and inflammatory cytokines released from activated macrophages in response to particulate debris greatly impact periprosthetic bone loss and consequent implant failure. In the present study, we found that a major polyphenolic component of green tea, (-)-epigallocatechin gallate (EGCG), inhibited Ti particle-induced TNF-alpha release in macrophages in vitro and calvarial osteolysis in vivo. The Ti stimulation of macrophages released TNF-alpha in a dose- and time-dependent manner, and EGCG substantially suppressed Ti particle-induced TNF-alpha release. Analysis of signaling pathway showed that EGCG inhibited the Ti-induced c-Jun N-terminus kinase (JNK) activation and inhibitory kappaB (IkappaB) degradation, and consequently the Ti-induced transcriptional activation of AP-1 and NF-kappaB. In a mouse calvarial osteolysis model, EGCG inhibited Ti particle-induced osteolysis in vivo by suppressing TNF-alpha expression and osteoclast formation. Therefore, EGCG may be a potential candidate compound for osteolysis prevention and treatment as well as aseptic loosening after total replacement arthroplasty.

Down-regulation of survivin suppresses uro-plasminogen activator through transcription factor JunB

Survivin, a member of the inhibitors of apoptosis protein family, is expressed during development and in various human cancers. However, the clinical relevance of survivin in cancer is still a matter of debate. Genes induced by hepatocyte growth factor (HGF) were screened using cDNA microarray technology in the stomach cancer cell lines, NUGC3 and MKN28. The levels of JunB, survivin, and uro-plasminogen activator (uPA) were up-regulated in cells treated with HGF in a dose-dependent manner. HGF-induced up regulation of JunB, survivin, and uPA was inhibited by pre-treatment with a MEK inhibitor (PD 98059). HGF-induced up-regulation of uPA was repressed by survivin knockdown. HGF enhanced the binding activity of JunB to the survivin promoter in control cells, but not in the JunB-shRNA cells. Transfection with survivin-shRNA resulted in a decrement of cell proliferation, as determined with MTT assays. In an in vitro invasion assay, significantly fewer cells transfected with survivin shRNA than control cells were able to invade across a Matrigel membrane barrier. In conclusion, survivin appeared to play an important role in the up-regulation of uPA induced by HGF via JunB and might contribute to HGF-mediated tumor invasion and metastasis, which may serve as a promising target for gastric cancer therapy.

Hint1 over expression inhibits the activity of AP-1 transcription factor in HepaG2 cells / 中华普通外科杂志

Objective To study the inhibition of AP-1 transcription factor activity by Hint1 gene over expression in HepG2 cell lines. Methods The Hintl gene was amplified, and then was inserted into the pcDNA3/HA eukaryotic expression plasmid. The constructed pHA-Hint1 plasmid was confirmed by DNA sequencing. The pHA-Hint1 was transfected into the HepG2 human hepatoma cells. Semi-quantitative RT-PCR and Western-blot were used to detecte the expression of HA-Hint1. The HepG2 cells were co-transfected with pHA-Hint1 and pAP-1/Luc luciferase reporter. At 36 h after transfection, luciferase assay system was used to detect the AP-1 transcription factor activity. Results The constructed pHA-Hint1 was confirmed by DNA sequencing, pHA-Hint1 gene transduction through lipofectine induced over-expression in HA-Hint1 mRNA (t =3.89, P<0.05) and HA-Hintl protein (t=3. 12, P<0.05). Co-transfection of Hint1 gene inhibits AP-1 luciferase activity. Cotransfection with increased concentration of a pHA-Hint1 plasmid (0 μg/ml, 0. 5 μg/ml, 1.0 μg/ml, 1.5 μg/ml, 2. 0 μg/ml) produced a concentration-dependent inhibition of AP-1 transcription factor activity. At the concentration of 1.5 μg/ml, and 2.0 μg/ml, the activity inhibition reaches significant difference ( F = 72. 009, P < 0. 05 ). Conclusion Over-expression of Hintl can, at least in part, inhibit the AP-1 transcription factor activity in HepG2 cells.

Aldosterone-induced TGF-beta1 Expression is Regulated by Mitogen-Activated Protein Kinases and Activator Protein-1 in Mesangial Cells

Aldosterone has been shown to stimulate renal TGF-beta1 expression. However, the mechanisms for aldosterone-induced TGF-beta1 expression have not been clearly determined in mesangial cells. We examined the role of extracellular-signal regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK) and activator protein- 1 (AP-1) in the aldosterone-induced TGF-beta1 expression in rat mesangial cells. TGF-beta1 protein in the conditioned medium released from rat mesangial cells was measured by sandwich ELISA, TGF-beta1 mRNA expression was analyzed by Northern blotting, AP-1 DNA binding activity was measured by EMSA and the ERK1/2, JNK activity was analyzed by western blotting. Aldosterone significantly stimulated TGF-beta1 protein production and TGF-beta1 mRNA expression in mesangial cells in a dose-dependent manner. Aldosterone significantly increased AP-1 DNA binding activity in mesangial cells. Pre-treatment of cells with AP-1 inhibitor, curcumin, blocked aldosterone-induced AP-1 DNA binding activity as well as aldosterone-induced TGF-beta1 production. Aldosterone increased phosphorylation of ERK1/2 and JNK in mesangial cells. Pre-treatment of cells with ERK1/2 inhibitor, PD98059, or JNK inhibitor, SP600125 significantly inhibited aldosterone-induced ERK1/2 and JNK activity and subsequently TGF-beta1 production, respectively. We conclude that aldosteroneinduced TGF-beta1 expression in mesangial cells is regulated by the ERK1/ 2, JNK and AP-1 intracellular signaling pathways.

Histone deacetylase inhibitor KBH-A42 inhibits cytokine production in RAW 264.7 macrophage cells and in vivo endotoxemia model

In light of the anti-inflammatory properties of histone deacetylase (HDAC) inhibitors, such as suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA), we examined a new HDAC inhibitor KBH-A42 for its anti-inflammatory activities. KBH-A42 showed noteworthy anti-inflammatory properties in vitro via suppression of the production of TNF-alpha, a proinflammatory cytokine, and nitric oxide (NO), a proinflammatory effector molecule, in LPS-stimulated RAW264.7 cells and peritoneal macrophages. It also inhibited TNF-alpha production in vivo as demonstrated in a LPS-induced mouse endotoxemia model. The levels of TNF-alpha, IL-1beta, IL-6 and iNOS mRNAs determined by RT-PCR propose that the inhibition of these pro-inflammatory mediators by KBH-A42 resulted from inhibiting expression of these genes. However, the EMSA study to see the effect of KBH-A42 on the binding of NF-kappaB, a transcription factor, to a specific DNA sequence showed that the binding of NF-kappaB to DNA was not changed regardless of increasing the concentration of KBH-A42 in the presence and absence of LPS stimulation. Interestingly, DNA binding of another transcription factor AP-1 dose-dependently increased by KBH-A42. KBH-A42 differentially regulated the phosphorylation of MAP kinases. While the phosphprylation of ERK1/2 and SAPK/JNK was not affected by KBH-A42, the phosphorylation of p38 decreased by KBH-A42. These results showed that KBH-A42 inhibits production of proinflammatory cytokines in macrophages by decreasing their mRNA levels, and p38 kinase is involved in the KBH-A42-mediated inhibition.

STP-A11, an oncoprotein of Herpesvirus saimiri augments both NF-kappaB and AP-1 transcription activity through TRAF6

Herpesvirus saimiri (HVS), a member of the gamma-herpesvirus family, encodes an oncoprotein called Saimiri Transforming Protein (STP) which is required for lymphoma induction in non-human primates. However, a detailed mechanism of STP-A11-induced oncogenesis has not been revealed yet. We first report that STP-A11 oncoprotein interacts with TNF-alpha receptor-associated factor (TRAF) 6 in vivo and in vitro. Mutagenesis analysis of the TRAF6-binding motif 10PQENDE15 in STP-A11 reveals that Glu (E)12 residue is critical for binding to TRAF6 and NF-kappaB activation. Interestingly, co-expression of E12A mutant, lack of TRAF6 binding, with cellular Src (Src) results in decreased transcriptional activity of Stat3 and AP-1, a novel target of STP-A11 compared to that of wild type. Furthermore, the presence of STP-A11 enhances the association of TRAF6 with Src and induces the translocation of both TRAF6 and Src to a nonionic detergent-insoluble fraction. Taken together, these studies suggest that STP-A11 oncoprotein up-regulates both NF-kappaB and AP-1 transcription activity through TRAF6, which would ultimately contribute cellular transformation.

Release of heat shock protein 70 (Hsp70) and the effects of extracellular Hsp70 on matric metalloproteinase-9 expression in human monocytic U937 cells

Heat shock protein 70 (Hsp70) release and its effects on pro-inflammatory cytokine production have been controversial. In this study, we investigated whether Hsp70 could be released from monocytes and activates matrix metalloproteinase-9 (MMP-9) gene expression. Hsp70 overexpression in human monocytic cell line U937 was found to increase PMA- induced MMP-9 expression and enhance cell motility. Hsp70 cDNA transfectants released Hsp70 protein into culture supernatants, and a part of released Hsp70 subsequently was bound to the surface of U937 cells. Addition of culture medium containing the extracelluar Hsp70 led to an increase not only in proMMP-9 secretion, but also the invasiveness of U937 cells through Matrigel or human umbilical vascular endothelial cells (HUVEC) in vitro. Immunodepletion of Hsp70 abolished its effect on MMP-9 expression. The released Hsp70 activated nuclear factor kappa B (NF-kappa B) and activating protein-1 (AP-1), which led to the activation of MMP-9 transcription. Taken together, these results suggest that extracellular Hsp70 induces the expression of MMP-9 gene through activation of NF-kappa B and AP-1.

Menin represses JunD transcriptional activity in protein kinase Ctheta-mediated Nur77 expression

TCR signaling leading to thymocyte apoptosis is mediated through the expression of the Nur77 family of orphan nuclear receptors. It has been shown that the Nur77 promoter is activated by at least two signaling pathways, one mediated by calcium and the other by protein kinase C (PKC). MEF2D has been known to regulate Nur77 expression in a calcium- dependent manner. The mechanism by which calcium regulates MEF2D is through dissociation of calcium-sensitive MEF2 corepressors (Cabin1/ HDACs, HDAC4/5) and the association with calcineurin-activated transcription factor NF-AT and the coactivator p300. However, little is known about how PKC activates the Nur77 promoter. Herein, we report that PKC theta targets AP-1 like response element in the Nur77 promoter where JunD constitutively binds. PKC theta triggers mitogen-activated protein kinase- inediated phosphorylation of JunD, and increases transcriptional activity of JunD, cooperatively with p300. Menin is identified as the transcriptional corepressor for JunD via recruitment of mSin3-istone deacetylases. In fact, Menin represses PKC theta/ p300-mediated transcriptional activity of JunD in T cell. Its dynamic regulation of histone modifiers with JunD is responsible for PKCq-synergistic effect on Nur77 expression in T cell.

Effects of AP-1 element on endothelin-1 gene transcription in homocysteine-induced HUVECs / 中国病理生理杂志

AIM:The objective of this study was to observe the effects of AP-1 element on endothelin-1(EDN1)gene transcription in homocysteine(Hcy)-induced human umbilical vein endothelial cells(HUVECs).METHODS:The redox level in Hcy-induced HUVECs was determined by using the fluorescent product DCF.The influences of Hcy on expressions of EDN1 mRNA and protein of c-jun/AP-1 in HUVECs were measured by the methods of RT-PCR and Western blotting,respectively.The EDN1 secretion level of HUVECs induced by Hcy was determined by enzymatic immunoassay.The transient transfection assay was performed and luciferase activity of transcriptional factor AP-1 reporter gene induced by Hcy was detected.RESULTS:The Hcy significantly increased EDN1 secretion,EDN1 mRNA expression and oxidative stress in HUVECs.Hcy remarkably induced the expression of pGL3-EDN1-AP-1(115/+135)luciferase reporter gene plasmid.However,basic expression of mutation of pGL3-EDN1-AP-1(-115/+135)luciferase reporter gene plasmid was downregulated markedly in HUVECs induced by Hcy.CONCLUSION:Redox-active and release of ROS are changed by Hcy.Activated AP-1 element plays an important role in EDN1gene transcription in HUVECs induced by Hcy.

Effects of Chlamydia pneumoniae infection and hyperlipidemia on the expression of NF-?B and AP-1 in myocardium / 中国病理生理杂志

AIM: To investigate the effects of Chlamydia pneumoniae infection and hyperlipidemia on the expression of NF-?B and AP-1 in the myocardium. METHODS: The indirect immunofluorescence method was used to examine wild C57BL/6J mice infected with Chlamydia pneumoniae and fed with an atherogenic diet. The expression of the subunit of NF-?B, P50, and c-Fos in the murine myocardium was observed. RESULTS: Chlamydia pneumoniae infection and hyperlipidemia induced the activation of NF-?B and AP-1 in murine myocardium. P50 and c-Fos were not detected in the controls, but there were different levels of positive expression in the experiments (P