Role of Dlg5/lp-dlg, a Membrane-Associated Guanylate Kinase Family Protein, in Epithelial-Mesenchymal Transition in LLc-PK1 Renal Epithelial Cells

Discs large homolog 5 (Dlg5) is a member of the membrane-associated guanylate kinase adaptor family of proteins, some of which are involved in the regulation of epithelial-to-mesenchymal transition (EMT). Dlg5 has been described as a susceptibility gene for Crohn's disease; however, the physiological function of Dlg5 is unknown. We show here that transforming growth factor-β (TGF-β)-induced EMT suppresses Dlg5 expression in LLc-PK1 cells. Depletion of Dlg5 expression by knockdown promoted the expression of the mesenchymal marker proteins, fibronectin and α-smooth muscle actin, and suppressed the expression of E-cadherin. In addition, activation of JNK and p38, which are stimulated by TGF-β, was enhanced by Dlg5 depletion. Furthermore, inhibition of the TGF-β receptor suppressed the effects of Dlg5 depletion. These observations suggest that Dlg5 is involved in the regulation of TGF-βreceptor-dependent signals and EMT

Effect of natural gas injection point on combustion and gasification efficiency of pulverized coal under blast furnace condition

The reduction of CO2 emission from the ironmaking process is an important issue from the view of environmental problems typified by global warming in recent years. Low RAR (reducing agent rate) operation of the blast furnace is one of effective methods for reducing CO2 emission. Injection of HRA (hydrogenous reducing agents) from the tuyere (where is the lower part of a blast furnace) is also effective measure. In this study, the influence of HRA injection point on combustion and gasification efficiency of pulverized coal (PC) in the case of simultaneous injection of HRA and PC from double-channel lance was examined by small scale combustion furnace and three-dimensional numerical simulation for permeability in the blast furnace. Combustion experimental conditions were in three cases, case 1: injected HRA from the outer side and PC from the inner side of double-channel lance, case 2: injected HRA from inner side and PC from outer side of double-channel lance and case 3: injected HRA and PC premixed. As a result, the combustion and gasification efficiency was increased in the order of case 1, case 2 and case 3. The rate of combustion and gasification of PC was investigated in case1. Not only the oxidation reaction was also accelerated CO2 and H2O gasification reaction in the case of simultaneous injection HRA and PC. A three-dimensional numerical simulation of the experimental furnace was conducted, we confirmed the increase of combustion temperature, the acceleration of oxygen consumption and gasification reaction as with the experimental results in the case of simultaneous injection HRA and PC

Effect of Natural Gas Injection Point on Combustion and Gasification Efficiency of Pulverized Coal under Blast Furnace Condition

The reduction of CO2 emission from the ironmaking process is an important issue from the view of environmental problems typified by global warming in recent years. Low RAR (reducing agent rate) operation of the blast furnace is one of effective methods for reducing CO2 emission. Injection of HRA (hydrogenous reducing agents) from the tuyere (where is the lower part of a blast furnace) is also effective measure. In this study, the influence of HRA injection point on combustion and gasification efficiency of pulverized coal (PC) in the case of simultaneous injection of HRA and PC from double-channel lance was examined by small scale combustion furnace and three-dimensional numerical simulation for permeability in the blast furnace. Combustion experimental conditions were in three cases, case 1: injected HRA from the outer side and PC from the inner side of double-channel lance, case 2: injected HRA from inner side and PC from outer side of double-channel lance and case 3: injected HRA and PC premixed. As a result, the combustion and gasification efficiency was increased in the order of case 1, case 2 and case 3. The rate of combustion and gasification of PC was investigated in case1. Not only the oxidation reaction was also accelerated CO2 and H2O gasification reaction in the case of simultaneous injection HRA and PC. A three-dimensional numerical simulation of the experimental furnace was conducted, we confirmed the increase of combustion temperature, the acceleration of oxygen consumption and gasification reaction as with the experimental results in the case of simultaneous injection HRA and PC

Dlg5 depletion disrupts epithelial cell morphology and induces the expression of mesenchymal marker proteins.

<p><b>A:</b> LLc-PK1 cells were transfected with Dlg5 siRNA (siDlg5#1: KD) or control siRNA and then incubated with 4 ng/ml of TGF-β for three days. The cells were lysed and immunoblotted using the indicated antibodies. Expression of β-tubulin was examined as a loading control. The upper band observed in immunoblotting with anti-Dlg5 is the larger variant of Dlg5. <b>B:</b> Cells were treated as in A and incubated for 60 hours. The cells were then photographed using phase contrast microscopy to examine cell morphology. <b>C:</b> Cells were transfected with Dlg5 siRNA or control siRNA and then immunostained using anti-Dlg5 or anti-E-cadherin antibodies. The asterisks indicate the cells that were transfected with Dlg5 siRNA. The scale bar indicates 10 µm. <b>D:</b> LLc-PK1 cells were transfected with Dlg5 siRNA or control siRNA and incubated for 24 hours. A GFP expression plasmid was then transfected into the cells with FLAG-Dlg5 or control plasmids. After incubation for two days, the cells were immunostained using an anti-SMA antibody. Fifty GFP-expressing cells were randomly selected, and the fluorescent intensity of SMA staining was examined. The graph shows the ratio of cells with higher SMA expression than background. <b>E:</b> LLc-PK1 cells were transfected with FLAG-Dlg5 or control plasmid and incubated for six hours. The cells were further transfected with Dlg5 siRNA or control siRNA. After two days of incubation, the cells were lysed and immunoblotted using the indicated antibodies. Arrows indicate endogenously expressed Dlg5, and an arrow head indicates FLAG-Dlg5. Dlg5 depletion induced the expression of mesenchymal marker proteins, and the re-expression of Dlg5 suppressed it.</p

Dlg5 depletion promotes JNK and p38 activation.

<p><b>A:</b> LLc-PK1 cells were stimulated with 4 ng/ml TGF-β and incubated for the indicated periods. Cells were then lysed and immunoblotted using the indicated antibodies. <b>B, C, D, E:</b> LLc-PK1 cells were transfected with Dlg5 siRNA (KD) or control siRNA and incubated for two days. Cell lysates were then immunoblotted using the indicated antibodies, and the results were quantitated. The values represent the mean ± S.E. from at least three independent experiments. Dlg5 depletion promoted JNK and p38 activation but not Smad2/3 activation.</p

TGF-β-mediated EMT decreases Dlg5 expression.

<p><b>A:</b> LLc-PK1 cells were incubated with 4 ng/ml of TGF-β for three days. Images were taken using phase contrast microscopy. TGF-β treatment induced morphological changes of LLc-PK1 cells. <b>B:</b> Three days after incubation with TGF-β, cells were lysed and immunoblotted using the indicated antibodies, which include an antibody for the epithelial marker E-cadherin, antibodies for the mesenchymal markers SMA and fibronectin, and antibodies for Dlg5 and β-catenin. As a loading control, vinculin expression was detected. <b>C:</b> LLc-PK1 cells were incubated with 4 ng/ml of TGF-β for three days. The cells were immunostained with anti-Dlg5 or anti-E-cadherin antibody. The scale bar indicates 10 µm. TGF-β treatment induced EMT and decreased Dlg5 expression. The results are representative of at least three independent experiments.</p

Dominant-negative mutants of JNK and p38 suppress the expression of mesenchymal marker proteins in Dlg5-depleted cells.

<p><b>A:</b> Six hours after transfection of a dominant-negative JNK mutant or a control plasmid into LLc-PK1 cells, the cells were further transfected with control or Dlg5 siRNA. After incubation for two days, expression of the indicated proteins was investigated by immunoblotting. <b>B:</b> Dominant-negative p38 mutant or a control plasmid was transfected into LLc-PK1 cells and treated and analyzed as in A. Expression of β-tubulin was detected as a loading control. The results are representative of at least three independent experiments. The expression levels of dominant-negative mutants were affected by Dlg5 knockdown by an unknown mechanism. Dominant-negative mutants of JNK and p38 suppressed the expression of fibronectin and SMA.</p

Inhibition of the TGF-β receptor suppresses the expression of mesenchymal marker proteins in Dlg5-depleted cells.

<p><b>A:</b> LLc-PK1 cells were transfected with Dlg5 siRNA (KD) or control siRNA and stimulated with or without 4 ng/ml TGF-β. Two days after the addition of 2 µM ALK5 inhibitor II (TGF-β receptor inhibitor), cells were lysed and immunoblotted using the indicated antibodies. <b>B:</b> LLc-PK1 cells were transfected with a Smad7 expression plasmid or control plasmid and incubated for six hours. The cells were then treated with or without TGF-β (left panel) or transfected with Dlg5 siRNA or control siRNA (right panel). Two days after further incubation, cells were lysed and the expression of the indicated proteins was investigated by immunoblotting. Expression of β-tubulin was detected as a loading control. The results are representative of at least three independent experiments. Both pharmacological and physiological inhibition of TGF-β receptor suppressed the expression of SMA and fibronectin induced in Dlg5-depleted cells.</p

Overexpression of Dlg5 attenuates the TGF-β-mediated increase in the expression of mesenchymal marker proteins.

<p>LLc-PK1 cells stably expressing GFP or GFP-tagged Dlg5 were treated with or without 4 ng/ml TGF-β. After two days of incubation, cells were lysed and protein expression was detected by immunoblotting using the indicated antibodies. Exogenously expressed Dlg5 suppressed the increase in SMA and fibronectin expression.</p