TGF-β facilitates Sp1/β-catenin interaction.

<p>Airway smooth muscle cells were stimulated with TGF-β (2 ng/ml) for 16 hours. Co-immunoprecipitation was performed as described in the Materials and Methods section. Immunocomplexes and whole cell extracts (WCE) were subjected to western analysis as indicated in the panels.</p

Sp1 is the transcription factor for TGF-β-induced WNT-5A expression in airway smooth muscle cells.

<p>(A-B) Mithramycin A attenuates WNT-5A mRNA and protein expression. (A) Cells were stimulated with TGF-β (2 ng/ml) in the presence or absence of Mithramycin A (300 nM) for 24 hours. WNT-5A mRNA was analyzed by qRT-PCR. Data represent mean ± SEM of 4 independent experiments. **p<0.01 compared to vehicle basal, ## p<0.01 compared to TGF-β-stimulated cells; 1-way ANOVA followed by Newman-Keuls multiple comparisons test. (B) Cells were stimulated with TGF-β (2 ng/ml) in the presence or absence of Mithramycin A (300 nM) for 48 hours. Whole cell extracts were prepared and WNT-5A protein abundance was evaluated by western analysis. GAPDH was assessed as loading control. (C, D) Cells were transfected with Sp1-specific or a non-targeting siRNA as control. Subsequently, cells were stimulated with TGF-β (2 ng/ml) for 24 hours and analyzed for the expression of Sp1 mRNA (C) and WNT-5A mRNA (D) by qRT-PCR. Data represent mean ± SEM of 5 independent experiments. *p<0.05, ***p<0.001 compared to non-targeting siRNA-transfected untreated control, #p<0.05, ### p<0.001 compared to non-targeting siRNA-transfected, TGF-β-stimulated cells; 1-way ANOVA followed by Newman-Keuls multiple comparisons test. (E) Mithramycin A attenuates TGF-β-induced extracellular matrix expression. Cells were stimulated with TGF-β (2 ng/ml) in the presence or absence of Mithramycin A (300 nM) for 24 hours. Collagen IαI and fibronectin mRNA was analyzed by qRT-PCR. Data represent mean ± SEM of 4 independent experiments. *p<0.05, **p<0.01 compared to vehicle basal, #p<0.05, ## p<0.01 compared to TGF-β-stimulated cells; 1-way ANOVA followed by Newman-Keuls multiple comparisons test. (F) Sp1 is recruited to WNT-5A promoter in response to TGF-β. Cells were left untreated or stimulated with TGF-β (2 ng/ml) for 16 hours. Chromatin was prepared and ChIP analysis was performed as described in the Materials and Methods section. PCR was carried out using primers specific for Sp1 binding region on <i>WNT-5A</i> promoter A after immunoprecipitation with anti-Sp1 or control IgG antibody. Input DNA from chromatin preparation before immunoprecipitation was amplified to ascertain the loading. Resulting PCR products were analyzed by DNA PAGE. (G) TAK1 mediates recruitment of Sp1 to <i>WNT-5A</i> promoter in response to TGF-β. Cells were left untreated or stimulated with TGF-β (2 ng/ml) in the presence or absence of LL-Z1640-2 (0.5 µM) for 16 hours. ChIP analysis was performed as described above.</p

TAK1 regulates total and active fraction of β-catenin in airway smooth muscle cells.

<p>(A-C) TAK1 signaling in total β-catenin regulation. Airway smooth muscle cells were either left unstimulated (vehicle basal) or stimulated with TGF-β (2 ng/ml) in the presence or absence of LL-Z1640-2 (0.5 µM), SB203580 (10 µM), SP600125 (10 µM) or the combination of SB203580 and SP600125 (10 µM each) for 24 hours. Whole cell extracts were subjected to western analysis for detection of total β-catenin protein abundance. GAPDH expression was examined as loading control. Graphs represent quantitation of band intensities for total β-catenin corrected for GAPDH as percentage of TGF-β-induced expression. Data represent mean ± SEM of 4-6 independent experiments. *p<0.05, **p<0.01 compared to vehicle basal, # p<0.05, ## p<0.01 compared to TGF-β-stimulated cells; 2-tailed Student's <i>t</i> test for paired observations. (D, E) Regulation of active β-catenin by TAK1. Airway smooth muscle cells were either left unstimulated (vehicle basal) or stimulated with TGF-β (2 ng/ml) in the presence or absence of LL-Z1640-2 (0.5 µM) for 16 or 24 hours as indicated. Whole cells extracts were subjected to western analysis for detection of active β-catenin protein abundance. Expression of GAPDH was assessed as loading control. Graphs represent quantitation of band intensities for active β-catenin corrected for loading control as percentage of TGF-β-induced expression. Data represent mean ± SEM of 5 independent experiments. *p<0.05, **p<0.01 compared to vehicle basal, # p<0.05, ## p<0.01 compared to TGF-β-stimulated cells; 2-tailed Student's <i>t</i> test for paired observations.</p

β-Catenin mediates TGF-β-induced WNT-5A expression in airway smooth muscle cells.

<p>(A) <i>De novo</i> protein synthesis is required for TGF-β-induced WNT-5A expression. Airway smooth muscle cells were either left unstimulated (vehicle basal) or stimulated with TGF-β (2 ng/ml) in the presence or absence of the protein synthesis inhibitor cycloheximide (5 µg/ml) for 24 hours. WNT-5A mRNA induction was evaluated by qRT-PCR. Data represent mean ± SEM of 4 independent experiments. **p<0.01, ***p<0.001 compared to vehicle basal, ## p<0.01 compared to TGF-β-stimulated cells; 2-tailed Student's <i>t</i> test for paired observations. (B-D) β-Catenin silencing reduces TGF-β-induced WNT-5A expression. Airway smooth muscle cells were transfected with β-catenin-specific siRNA or a non-targeting siRNA as control. Subsequently, cells were stimulated with TGF-β (2 ng/ml) for 24 hours (mRNA; B,C) or 48 hours (protein; D). (B,C) Expression of β-catenin mRNA (B) and WNT-5A mRNA (C) was determined by qRT-PCR and expressed relative to non-targeting siRNA transfected, untreated control. Data represent mean ± SEM of 5 independent experiments. *p<0.05, **p<0.01 compared to non-targeting siRNA-transfected, untreated control, # p<0.05, ### p<0.001 compared to non-targeting siRNA-transfected, TGF-β-stimulated cells; 2-tailed Student's <i>t</i> test for paired observations. (D) Western blot analysis was performed to analyze WNT-5A and β-catenin protein expression in whole cell extracts. Equal protein loading was verified by the analysis of GAPDH. (E) Forced increase in β-catenin abundance elevates WNT-5A protein level. Cells were transfected with S33Y-β-catenin mutant or a GFP expression vector as control. Subsequently, cells were either left untreated or stimulated with TGF-β (2 ng/ml) for 48 hours. Western blot analysis was performed to determine the abundance of WNT-5A and total β-catenin at protein level. GAPDH expression assessed as loading control. (F) Canonical WNT ligand stimulation increases WNT-5A gene expression. Cells were stimulated with L-cells-derived WNT-3A conditioned medium or control conditioned medium for 24 hours. Expression of WNT-5A mRNA was evaluated by qRT-PCR and expressed relative to control conditioned medium. Data represent mean ± SEM of 5 independent experiments. **p<0.01 compared to control conditioned medium; 2-tailed Student's <i>t</i> test for paired observations.</p

TAK1-activated p38/JNK signaling regulates WNT-5A induction in airway smooth muscle cells.

<p>(A) TAK1 activates p38 and JNK. Airway smooth muscle cells were stimulated with TGF-β (2 ng/ml) in the presence or absence of LL-Z1640-2 (0.5 µM) for 30 and 60 minutes. Whole cells extracts were immunoblotted for phospho-p38 and phospho-JNK using specific antibodies. Equal protein loading was verified by the analysis of β-actin. (B–D) p38 and JNK involvement in WNT-5A expression. Airway smooth muscle cells were stimulated with TGF-β (2 ng/ml) in the presence or absence of SB203580 (10 µM) or SP600125 (10 µM) or combination of both SB203580 and SP600125 (10 µM each) for 24 hours. RNA was isolated and WNT-5A mRNA expression was determined by qRT-PCR, corrected for 18S rRNA and expressed relative to vehicle basal. Data represent mean ± SEM of 4–6 independent experiments. **p<0.01, ***p<0.001 compared to vehicle basal, ### p<0.001 compared to TGF-β-stimulated cells; 1-way ANOVA followed by Newman-Keuls multiple comparisons test.</p

8-pCPT-2′-<i>O</i>-Me-cAMP prevents CSE-induced breakdown of IκBα and p65 nuclear translocation.

<p>p65 nuclear translocation was determined by immunofluorescence using p65 antibodies on hTERT-ASM cells stimulated without (control) or with 15% CSE for 2 hrs, alone or in combination with 100 µM 8-pCPT-2′-<i>O</i>-Me-cAMP or 500 µM 6-Bnz-cAMP. Representative results of 3 separate experiments are shown (A) with the quantification of p65 nuclear staining (B). hTERT-ASM cells were treated with 15% CSE, 100 µM 8-pCPT-2′-<i>O</i>-Me-cAMP, 500 µM 6-Bnz-cAMP or their combinations for 1 hr. Cells were lysed and IκBα levels were determined by Western blot analysis (C). Bands were normalized to GAPDH. Representative immunoblots of IκBα and GAPDH are shown. Data are presented as means±SEM of 6–7 separate experiments. Statistical analysis was performed by one-way ANOVA followed by a Dunnett post-hoc test. ^*<i>P</i><0.05, ^***<i>P</i><0.001 compared to basal control. ^#<i>P</i><0.05, ^###<i>P</i><0.001 compared to CSE.</p

Fenoterol, 6-Bnz-cAMP and 8-pPCT-2′-<i>O</i>-Me-cAMP reduce CSE-induced IL-8 release from human ASM cells.

<p>hTERT ASM cells (A, B and C) and human primary ASM cells (F) were stimulated with 15% CSE in the absence or presence of fenoterol (0.1 or 1 µM), 6-Bnz-cAMP (100 µM or 500 µM) or 8-pCPT-2′-<i>O</i>-Me-cAMP (30 or 100 µM) for 24 hrs. Basal IL-8 release was 109,4±33,2 pg/ml in hTERT ASM cells and 7,5±4,7 in primary human ASM cells. Cells stimulated with 15% CSE for 6 hrs showed an increase in IL-8 mRNA expression which was reduced by fenoterol (1 µM), 6-Bnz-cAMP (500 µM) or 8-pCPT-2′-O-Me-cAMP (100 µM) (E). CSE, Fenoterol, 6-Bnz-cAMP and 8-pPCT-2′-<i>O</i>-Me-cAMP did not affect cell viability (D). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031574#s2" target="_blank">Results</a> are represented as means ± SEM of 6–22 separate experiments. Statistical analysis was performed by one-way ANOVA followed by a Newman-Keuls post-hoc test. ^***<i>P</i><0.001,^**<i>P</i><0.01 compared to basal control; ^#<i>P</i><0.05, ^##<i>P</i><0.01. ^###<i>P</i><0.001 compared to CSE-stimulated control.</p

Epac1 is down-regulated by CSE.

<p>hTERT-ASM or primary ASM cells were treated for 4 and 24 hrs with 15% CSE. Then, cells were lysed for protein and mRNA determination. mRNA (A) and protein expression (C) of Epac1 was significant down regulated after exposure to CSE, while Epac2 was unaffected in hTERT cells. Same results were obtained in primary cells (E) Catalytic (PKA-C) and regulatory type II (PKA-RII) subunits of PKA mRNA (B & F) and protein (D) expression were not affected by CSE exposure in hTERT cells and in primary ASM cells. Protein expression of Epac1 and Epac2 (C) and PKA-C PKA-RII (D) were normalized to β-actin (for Epac) and GAPDH (for PKA). mRNA expression was normalized to 18 S. Data represent mean±SEM of 3–5 independent experiments. Statistical analysis was performed by one-way ANOVA followed by a Newman-Keuls post-hoc test. ^*<i>P</i><0.05 compared to time point 0 hrs.</p

Epac and PKA expression in COPD patients.

<p>Expression of PKA-C and PKA-RII (A) and Epac1 and Epac2 (B) was evaluated by immunoblotting. Equal protein loading was verified by the analysis of β-actin. Responses were quantified by densitometry and normalized to the expression of β-actin. Data are derived from 9 controls and 15–19 COPD patients. Median of each group is indicated by -----. *P<0.05. Statistical differences between control and COPD were determined by non-parametric Mann-Whitney test.</p

Inhibition of PKA attenuates the effect of 6-Bnz-cAMP on CSE-induced IL-8 release.

<p>Phosphorylation of VASP in hTERT-ASM cells treated with 100 µM 8-pCPT-2′-<i>O</i>-Me-cAMP or 500 µM 6-Bnz-cAMP in the absence or presence of the PKA inhibitor H89 (300 nM) was analysed by using an antibody, which recognizes both the phosphorylated VASP (phospho-VASP) and the non phosphorylated VASP (VASP) (A). VASP was normalized to GAPDH. Representative immunoblots of 3 experiments are shown. hTERT-ASM were pre-treated without (white bars) or with (black bars) 300 nM H89 (B) or 500 µM of both Rp-8-Br-cAMPS and Rp-cAMPS (C) for 30 min before stimulation with 15% CSE, 100 µM 8-pCPT-2′-<i>O</i>-Me-cAMP, 500 µM 6-Bnz-cAMP or their combinations. Data are presented as means±SEM of 3–9 separate experiments. Statistical analysis was performed by one-way ANOVA followed by a Newman-Keuls post-hoc test. ^***<i>P</i><0.001 compared to basal control. ^#<i>P</i><0.05, ^###<i>P</i><0.001 compared to CSE. ^‡<i>P</i><0.05, ^‡‡‡<i>P</i><0.001.</p