Blocking p38 MAPK inhibits myofibroblast proliferation.

<p><b><i>A</i></b>, IMR-90 cells stably expressing a p38 MAPK dominant negative (pcDNA-p38KM) and cells stably transfected with an empty vector (pcDNA) were serum-starved for 24 h and treated with/without TGF- β1 (2 ng/ml) for 48 h followed by stimulation with 10% fetal bovine serum for 24 h (n = 6 per group), data shown as mean±S.E.M. *indicates <i>p</i> < 0.05 vs. control pcDNA “fibroblasts”. **indicates <i>p</i> < 0.01 vs. control pcDNA “myofibroblasts” (TGF-β1 pre-treated). Similar results were obtained from 3 independent experiments. <b><i>B</i></b>, Cells described in (A) were grown in 96-well plates and serum-starved for 24 h and treated with/without TGF- β1 (2 ng/ml) for 48 h followed by BrdU labeling for 24 h in the presence of 10% fetal bovine serum (n = 6 per group), data shown as mean±S.E.M. *indicates <i>p</i> < 0.05 vs. control pcDNA “fibroblasts”. **indicates <i>p</i> < 0.01 vs. control pcDNA “myofibroblasts” (TGF-β1 pre-treated). Similar results were obtained from 3 independent experiments. <b><i>C</i></b>, IMR-90 cells stably expressing SMAD2 <i>sh</i>RNA and cells stably transfected with an empty vector (pSU6H) were serum-starved for 24 h and treated with/without TGF- β1 (2 ng/ml) for 48 h followed by stimulation with 10% fetal bovine serum for 24 h (n = 6 per group) data shown as mean±S.E.M. *indicates <i>p</i> < 0.05 vs. control pSU6H “fibroblasts” (no TGF-β1 pre-treatment). **indicates <i>p</i> < 0.05 vs. control pcDNA “myofibroblasts” (TGF-β1 pre-treated). <b><i>D</i></b>, Cells described in (C) were grown in 96-well plates and serum-starved for 24 h and treated with/without TGF- β1 (2 ng/ml) for 48 h followed by BrdU labeling for 24 h in the presence of 10% fetal bovine serum (n = 6 per group) data shown as mean±S.E.M. *indicates <i>p</i> < 0.05 vs. control pSU6H “fibroblasts” (no TGF-β1 pre-treatment). **indicates <i>p</i> < 0.05 vs. control pcDNA “myofibroblasts” (TGF-β1 pre-treated).</p

<i>sh</i>RNA knock-down of Cav-1 enhances fibroblast/myofibroblast proliferation and protects against serum deprivation-induced apoptosis.

<p><b><i>A</i></b>, IMR-90 cells stably transfected with a plasmid encoding <i>sh</i>RNA targeted against Cav-1 (pSU6H-<i>sh</i>Cav1) or with control plasmid (pSU6H) were treated with/without TGF-β1 (2 ng/ml) for 24 h. Cell lysates were obtained and Western blots for Cav-1, α-smooth muscle actin (α-SMA) and β-tubulin performed. <b><i>B</i></b>, Stably-transfected cells described in (A) were serum-deprived for 24 h and treated with/without TGF-β1 (2 ng/ml) for 48 h followed by stimulation with 10% FBS for 24 h. Cell counts were assessed both prior to and after serum stimulation with an automated Coulter counter (n = 6 per group) shown as mean±S.E.M. *indicates <i>p</i> < 0.05 vs. control pSU6H “fibroblasts”. **indicates <i>p</i> < 0.05 vs. pSU6H “myofibroblasts” (TGF-β1 pre-treated). Similar results were obtained from 3 independent experiments. <b><i>C</i></b>, Stably transfected cells described in (A) were serum-deprived for 24 h and treated with/without TGF-β1 (2 ng/ml) for 48 h followed by BrdU labeling for 24 h in the presence of 10% FBS (n = 6 per group) shown as mean±S.E.M. *indicates <i>p</i> < 0.05 vs. control pSU6H “fibroblasts”. Similar results were obtained from 3 independent experiments. <b><i>D</i></b>, Quiescent stably-transfected cells described in (<b><i>A</i></b>) were treated with/without TGF-β1 (2 ng/ml) for 5 days and apoptotic assay for <i>ss</i>DNA performed as described in “Methods” (n = 6 per group) shown as mean±S.E.M. *indicates <i>p</i> < 0.05 vs. control pSU6H “fibroblasts”. **indicates <i>p</i> < 0.05 vs. pSU6H “myofibroblasts” (TGF-β1 pre-treated). Similar results were obtained from 3 independent experiments.</p

Down-regulation of Cav-1 by TGF-β1 is mediated by p38 MAPK-dependent and SMAD-independent mechanisms.

<p><b><i>A</i></b>, IMR-90 cells were treated with inhibitors of p38 MAPK (SB203580; 6 μM) or ALK5 (SB431542; 0.5 μM) for 30 min prior to treatment with or without TGF-β1 (2 ng/ml) for a period of 48 h. Cell lysates were extracted and Western immunoblotting performed with an antibody against Cav-1; the blot was then stripped and probed for β-tubulin. <b><i>B</i></b>, Densitometric analyses of blots in (A) showed as % inhibition of baseline Cav-1 protein expression levels treated with TGF-β1. *indicates effect of SB203580 to completely block the inhibitory effect of TGF-β1 on Cav-1 expression. Results are averages of at least three independent experiments. Data are presented as mean±S.E.M. <b><i>C</i></b>, IMR-90 cells stably transfected with a kinase-deficient p38 MAPK (pcDNA-p38KM) or control vector (pcDNA) were treated with or without TGF-β1 (2 ng/ml) for 24 h. Cell lysates were obtained and subjected to SDS-PAGE and immunoblotted for Cav-1 and α-smooth muscle actin (α-SMA); blots were stripped and probed for β-tubulin. <b><i>D</i></b>, IMR-90 cells stably expressing SMAD2 <i>sh</i>RNA (pSU6H-<i>sh</i>SMAD2) or control vector (pSU6H) were treated with/without TGF-β1 (2 ng/ml) for 24 h. Cell lysates were immunoblotted for SMAD2, Cav-1 and α-SMA. The blots were stripped and probed for β-tubulin.</p

Schematic representation of TGF-β1-activated signaling pathways mediating mesenchymal cell growth -suppressive and -promoting effects.

<p>TGF-β1 activates the cell surface TGF-β receptor(s) complex that leads to rapid activation of the canonical SMAD pathway as well as the SMAD-<i>in</i>dependent p38 MAPK pathway. Activation of the SMAD pathway is required for the induction of a cellular program of growth-arrest and myofibroblast differentiation. In contrast, activation of the p38 MAPK pathway, independently of SMAD2/3, is required the down-regulation of Cav-1 by TGF-β1. Down-regulation of Cav-1 by TGF-β1 “primes” differentiated myofibroblasts for enhanced proliferative responses to mitogens and resistance to apoptosis. These divergent TGF-β signaling pathways may explain, in part, the contextual effects of TGF-β1 as both a growth-inhibitor and –promoter on the same target (mesenchymal) cells.</p

HO-1 protects CE-induced injury, permeability increase and apoptosis.

<p>HO-1 WT and HO-1 KO mice were exposed to either 20 μl of CE or held as controls for 24 hours. (A) The lung tissues were stained with H&E and images under a laser scanning microscopy. Data shown are representative of three donors. (B) Lung inflammation was detected by BAL cell counts. (C) Lung permeability was evaluated by BAL protein content. (D) The quantitative results from TUNEL assay were presented as percentage of TUNEL positive cells per field. Data are shown as a mean ± SD from three different experiments. * <i>p</i> < 0.05, ** <i>p</i> < 0.01 vs HO-1 WT (-) CE; ## <i>p</i> < 0.01 vs HO-1 KO (-) CE; && <i>p</i> < 0.01 vs HO-1 WT (+) CE by a one-way ANOVA with HSD test.</p

Morphological changes, reduction in cell diameter, permeability increase and disruption of intercellular junctions induced by CE.

<p>(A) Gills of zebrafish were exposed to either CE (150 ppm) or held as controls for 24 hours or 56 hours and stained with H&E. Digital micrographs were obtained at 20 x magnifications. Arrows point to edema and blebbing of gill epithelium. The ratios of gill area/gill length were calculated using Image Software (NIH, Bethesda, MD, USA) and presented as 1-dimensional area measurements. Data is quantified and shown as mean ± SD of three independent experiments. ** <i>p</i> < 0.01 vs control by a one-way ANOVA with HSD test. (B) Cell diameter measurements. BEAS-2B cells were grown to confluence in 65 mm dishes and exposed to 0 to 150 ppm of CE for 2 hours (n = 3). Data are shown as a mean ± SD. * <i>p</i> < 0.05 and ** <i>p</i> < 0.01 vs control by a one-way ANOVA with HSD test. (C) Permeability measurement of the bronchial epithelium of the airway. The sub-acute response to CE exposure was modeled by ECIS. BEAS-2B cells were seeded into the ECIS array. Cells were allowed to cover the gold electrodes in each well of the array prior to exposure to CE (0 ppm to 70 ppm). Real-time measurements of the electric resistance of the bronchial epithelial monolayers were obtained at 64 kHz. Resistance measurements were normalized with respect to the values in each well 1 hour prior to the initiation of the exposure. This time period corresponded to 16 hours after the seeding of the cells and was designated as t = 0 hour in the graph. The data are representative of three independent experiments. (D) BREA-2B cells were culture with or without 100 ppm CE for 1 hour. Protein expression of ZO-1 and actin filaments was detected using immunofluorescence microscopy (original magnification, ×40) with rabbit anti-ZO-1 (green) and phalloidin (red). Representative images captured from BEAS-2B cells are shown.</p

CE-induced apoptosis is caspase-3 dependent.

<p>(A) CE instigates apoptosis in BEAS-2B cells. Cells were pretreated with or without 10 μM ZnPP for overnight. Following exposure of BEAS-2B cells to 0 ppm, 150 ppm or 300 ppm (data not shown) of CE for 1 or 4 hours, flow cytometry dot plots for the simultaneous binding of Annexin V-FITC and PI uptake were shown. Numbers in the gates represent percentages of Dead (D) cells, as well as early (E), and late (L) apoptotic events. The data are representative of three independent experiments. Percentages of dead cells (PI⁺Annexin V^-) and apoptotic cells (Annexin V⁺) were quantified and data are shown as a mean ± SD. * <i>p</i> < 0.05, ** <i>p</i> < 0.01 vs no CE control in the absence of ZnPP; # <i>p</i> < 0.05, ## <i>p</i> < 0.01 vs no CE treatment in the presence of ZnPP; & <i>p</i> < 0.05, && <i>p</i> < 0.01 vs with CE treatment in the absence of ZnPP by a one-way ANOVA with HSD test. (B) Representative western blots and associated quantification for active caspase-3, normalized by β-actin content. BEAS-2B cells were exposed to 0 to 150 ppm of CE for 4 hours. Antibodies specific cleaved caspase-3 was used and β-actin was used as a loading control. The representative blots from three independent experiments are shown. The densities of protein bands were determined by densitometry and the data represent a one-fold increase from the control density. (C) Caspase-3 activity was measured using a DEVD-pNA calorimetric assay. After treatment with different concentration of CE for 4 hours, cells were lysed and 100 μg of protein was incubated with 200 μM DEVD-pNA for 6 hours at 37°C. Absorbance measurements were taken at a wavelength of 405 nm and the fold induction of caspase-3 activity relative to the control was shown. * <i>p</i> < 0.05 and ** <i>p</i> < 0.01 vs control by a one-way ANOVA with HSD test. (D) Mice tracheal explants were isolated and IHC analysis was performed after exposure 0 ppm or 150 ppm CE for 2 hours. (E) Blue crabs were exposed to 0 ppm or 150 ppm CE for 19 hours. Gill tissues were harvested for IHC analysis using a cleaved caspase-3 polyclonal antibody (1:800 dilution) followed by treatment with biotinylated goat anti-rabbit antibody and streptavidin–alkaline phosphatase (AP), which produced a red coloration for cleaved caspase-3 positive areas.</p

Upregulation of HO-1 and NOX4 in response to CE stimulation.

<p>Zebrafish were exposed to either CE 150 ppm or control for 56 hours and IHC analysis was performed for HO-1 (A) and NOX4 (B) expression. CE exposure is accomplished by holding adult zebrafish in 1 liter borosilicate class beakers (acid washed, followed with neutralizing alkali) to eliminate any potential reaction of the CE with plastic. (C) Cell lysates from BEAS-2B cells were analyzed by western blotting with anti-HO-1 and NOX4 antibodies at different concentrations of CE for 4 hours. β-actin was used as loading control. For quantification of the HO-1 and NOX4 expression, membranes were scanned and the bar graphs illustrated the relative expression of HO-1 and NOX4 by densitometry. The signal intensity for HO-1 or NOX4 at control was set to 1.0. (D) BEAS-2B cells were exposed to either CE or control for 4 hours, and HO activity was measured as pmol of bilirubin formed per mg protein per h. Data presents mean values of three independent experiments. Data are shown as a mean ± SD. ** <i>p</i> < 0.01 vs control by a one-way ANOVA with HSD test.</p

Proposed schematic: HO-1 contributes to protection of epithelium from CE-induced injury, ROS and apoptosis.

<p>CE exposure triggers oxidative stress, which induced cleavage of ZO-1, ZO-2, occludin, F-actin, E-cadherin, FAK cleavage, permeability increase, and activation of CRP, NOX4 and HO-1. ROS production and subsequent caspase-3 dependent apoptosis attribute to the induction of both CRP and NOX4. HO-1 and/or CORM-2 are in charge of the protection of CE-induced injury, ROS generation and apoptosis.</p

HO-1 stabilizes the adhesion proteins E-cadherin and FAK.

<p>(A) HO-1 WT and HO-KO mice were exposed to either 20 μl of CE or held as controls for 24 hours. Cell lysates were prepared from lung epithelial cells and analyzed by western blotting with anti-E-cadherin and FAK antibodies. (B) The densities of protein bands were determined by densitometry and the data represent a one-fold increase from the control density. Data are shown as a mean ± SD from three different experiments. * <i>p</i> < 0.05, ** <i>p</i> < 0.01 vs HO-1 WT (-) CE; ## <i>p</i> < 0.01 vs HO-1 KO (-) CE; && <i>p</i> < 0.01 vs HO-1 WT (+) CE by a one-way ANOVA with HSD test.</p