Proteasome Inhibition Augments Cigarette Smoke-Induced GM-CSF Expression in Trophoblast Cells via the Epidermal Growth Factor Receptor

Maternal cigarette smoking has adverse effects on pregnancy outcomes. The granulocyte-macrophage colony-stimulating factor (GM-CSF) is an essential cytokine for a normal pregnancy. We investigated the impact of cigarette smoke extract (CSE) on GM-CSF expression in human cytotrophoblast cells and suggested a cellular mechanism underlying the CSE-induced GM-CSF expression. An immortalized normal human trophoblast cell line (B6Tert-1) was treated with CSE. The viability and proliferation of the CSE-treated B6Tert-1 cells were evaluated, and the expression of GM-CSF in these cells was quantified at the mRNA and the protein levels by means of reverse-transcription and quantitative polymerase chain reaction (RT-qPCR); and enzyme-linked immunosorbent assay (ELISA), respectively. Human trophoblast cells treated with CSE had an increased expression of GM-CSF at both the mRNA and the protein levels. The CSE-induced GM-CSF expression was synergistically enhanced by the addition of the proteasome inhibitor MG-132, but inhibited by AG-1478, an inhibitor of the epidermal growth factor receptor (EGFR) kinase. Furthermore, CSE treatment increased the phosphorylation of the extracellular-signal regulated kinases (ERK1/2) in the trophoblast cells. The expression of other growth factors such as heparin-binding epidermal growth factor-like growth factor (HB-EGF) and vascular endothelial growth factor (VEGF) was also evaluated. Our data suggested that cigarette smoking and proteasome inhibition synergistically up-regulate GM-CSF cytokine expression by activating the EGFR signaling pathway

Proteasome inhibition augments cigarette smoke-induced GM-CSF expression in trophoblast cells via the epidermal growth factor receptor.

Maternal cigarette smoking has adverse effects on pregnancy outcomes. The granulocyte-macrophage colony-stimulating factor (GM-CSF) is an essential cytokine for a normal pregnancy. We investigated the impact of cigarette smoke extract (CSE) on GM-CSF expression in human cytotrophoblast cells and suggested a cellular mechanism underlying the CSE-induced GM-CSF expression. An immortalized normal human trophoblast cell line (B6Tert-1) was treated with CSE. The viability and proliferation of the CSE-treated B6Tert-1 cells were evaluated, and the expression of GM-CSF in these cells was quantified at the mRNA and the protein levels by means of reverse-transcription and quantitative polymerase chain reaction (RT-qPCR); and enzyme-linked immunosorbent assay (ELISA), respectively. Human trophoblast cells treated with CSE had an increased expression of GM-CSF at both the mRNA and the protein levels. The CSE-induced GM-CSF expression was synergistically enhanced by the addition of the proteasome inhibitor MG-132, but inhibited by AG-1478, an inhibitor of the epidermal growth factor receptor (EGFR) kinase. Furthermore, CSE treatment increased the phosphorylation of the extracellular-signal regulated kinases (ERK1/2) in the trophoblast cells. The expression of other growth factors such as heparin-binding epidermal growth factor-like growth factor (HB-EGF) and vascular endothelial growth factor (VEGF) was also evaluated. Our data suggested that cigarette smoking and proteasome inhibition synergistically up-regulate GM-CSF cytokine expression by activating the EGFR signaling pathway

GM-CSF and EGF increase viability and proliferation of B6Tert-1 cells.

<p>B6Tert-1 cells were seeded in 96-well plates and treated with GM-CSF or EGF at different concentrations as indicated in FD medium for 24 h. The data are expressed as the percentage of treated/untreated. Each data point represents mean ± SEM (n = 7).</p

Effects of inhibitors on CSE-induced GM-CSF mRNA expression.

<p>(A) Changes of GM-CSF mRNA expression level in B6Tert-1 cells treated with different agents in FD medium. CSE: 10% cigarette smoke extract; MG-132: proteasome inhibitor at 5 µM; AG-1478: EGFR kinase inhibitor at 5 µM; U0126: MEK inhibitor at 5 µM. Cells were pre-treated with inhibitor(s) for 30 min, and then with 10% CSE for another 5 h. DMSO was used as a vehicle control. The asterisk (*) indicates a statistically significant difference (<i>p</i><0.05) when compared with CSE-treated cells. (B) Western blot analysis of the phosphorylation state of ERK1/2 in B6Tert-1 cells treated with 10% CSE without or with inhibitors (5 µM each) as indicated for 30 min. Total ERK1/2 and GAPDH were used as the total protein and loading controls. Lane 1: FD (no treatment); lane 2: 10% CSE; lane 3: 10% CSE/MG-132; lane 4: 10% CSE/MG-132 and AG-1478; lane 5: MG-132 alone. The image represents one of three independently performed experiments. (C) Immunofluorescent staining showing the cellular distribution of NF-κB p65 subunit in B6Tert-1 cells under different treatment conditions. a: FD alone; b: 10% CSE; c: 10% CSE/MG-132; d: MG-132 alone; e: TNF-α: 50 ng/ml; and f: TNF-α/MG-132. “N” indicates the nucleus and the arrow indicates the NF-κB p65 subunit staining. Magnification: 20×10. (D) Western blot analysis of the distribution of NF-κB p65 subunit in B6Tert-1 cells under different treatment conditions. Cytoplasmic proteins were blotted with antibodies against NF-κB and GAPDH while nuclear proteins were blotted with antibodies against NF-κB and nucleoporin p62. Lanes 1: FD (no treatment); lanes 2: 10% CSE; lanes 3: 10% CSE/MG-132; and lanes 4: MG-132 alone. The image represents one of three independently performed experiments.</p

Cigarette smoke extract increases GM-CSF expression in B6Tert-1 cells.

<p>(A) Bar graph of real-time RT-qPCR data of GM-CSF mRNA expression in B6Tert-1 cells treated with 10% CSE in growth medium for 2 days. The relative GM-CSF mRNA expression level was determined against the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA level. Data are mean ± SEM. The asterisk (*) indicates a statistically significant difference (<i>p</i><0.05), when compared with the control (untreated) cells. (B) Bar graph of GM-CSF ELISA data of the secreted GM-CSF in B6Tert-1 conditioned medium. The medium was collected after 2 days of exposure to 10% CSE. The asterisk (*) indicates a statistically significant difference (<i>p</i><0.05), when compared with the control (untreated) cells. Data are mean ± SEM.</p

Effects of CSE and proteasome inhibition on VEGF and HB-EGF mRNA expression.

<p>Cells were pre-treated with inhibitor(s) for 30 min, and then with 10% CSE for another 5 h. DMSO was used as a vehicle control. A: VEGF mRNA expression levels in B6Tert-1 cells treated with different agents in FD medium. B: HB-EGF mRNA expression levels in B6Tert-1 cells treated with different agents in FD medium. CSE: 10% cigarette smoke extract; MG-132: proteasome inhibitor at 5 µM; AG-1478: EGFR kinase inhibitor at 5 µM. The asterisk (*) indicates a statistically significant difference (<i>p</i><0.05) when compared with CSE-treated cells.</p

EGF up-regulates GM-CSF mRNA expression in B6Tert-1 cells.

<p>(A) Bar graph of real-time RT-qPCR data of GM-CSF mRNA expression in B6Tert-1 cells treated with EGF. FD: untreated; EGF: 50 ng/ml; AG-1478: 5 µM. Cells were pre-treated with AG-1478 for 30 min and then with 10% CSE for another 5 h. The asterisk (*) indicates a statistically significant difference (<i>p</i><0.05) when compared with the untreated (FD) cells.</p

Viability and proliferation assays.

<p>B6Tert-1 cells (1×10⁴) were seeded in a 96-well plate in triplicates and grown overnight. Cigarette smoke extract (CSE) was added in FD medium at different final concentrations as indicated, and the cells were incubated for another 24 h. The viability and proliferation rate were monitored as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043042#s4" target="_blank">Materials and Methods</a>. The data are expressed as the percentage of CSE-treated/untreated, and represent the mean ± SEM. The experiment was repeated for three times.</p