Effect of IL-1β on CRE-dependent gene expression in human airway smooth muscle cells

Abstract

caused a marked increase in CRE-binding protein (CREB) phosphorylation, which was attenuated by IL-1␤ (2 ng/ml). This effect of IL-1␤ was abolished by the cyclooxygenase (COX) inhibitor indomethacin. To examine CRE-driven gene expression, we transiently transfected HASM cells with a construct containing CRE upstream of a luciferase reporter gene. ISO (6 h) caused a sixfold increase in luciferase activity. IL-1␤ (24 h) alone also increased luciferase activity, although to a lesser extent (2-fold). However, the ability of ISO to elicit luciferase expression was markedly reduced in cells treated with IL-1␤. Indomethacin, the MEK and p38 inhibitors U-0126 and SB-203580, the protein kinase A inhibitor H-89, and dexamethasone each completely abolished the ability of IL-1␤ to induce CRE-driven gene expression but only slightly increased the ability of ISO to induce CRE-driven gene expression in IL-1␤-treated cells. IL-1␤ also attenuated dibutyryl cAMP-induced CRE-driven gene expression, but not dibutyryl cAMP-induced CREB phosphorylation. Tumor necrosis factor-␣ (10 ng/ml) also attenuated ISO-induced CRE-driven gene expression, even though it was without effect on ISO-induced cAMP formation or ISO-induced CREB phosphorylation. The results suggest that IL-1␤ and tumor necrosis factor-␣ may attenuate the ability of ␤-agonists to induce expression of genes with CRE in their regulatory regions at least in part through events downstream of CREB phosphorylation. mitogen-activated protein kinase; cyclooxygenase-2; dexamethasone; cAMP response element binding; luciferase; tumor necrosis factor-␣

Selected Contribution: TNF-α modulates murine tracheal rings responsiveness to G-protein-coupled receptor agonists and KCl

“…Cultured murine ASM cells may represent an interesting approach to investigate the mechanisms by which TNF-␣ modulated ␤-adrenergic receptor function, but unfortunately, such an in vitro model is not available. Previous studies using cultured human ASM cells provide important observations such as the ability of TNF-␣ to decrease isoproterenol-induced adenylyl cyclase activity (19) and cAMP-dependent gene expression (36). Here we found that TNF-␣ did not attenuate the relaxant response induced by PGE 2 or forskolin, a nonreceptor-dependent relaxant agent that directly activates Fig.…”

Oncostatin M causes VEGF release from human airway smooth muscle: synergy with IL-1β

“…We have previously reported that OSM is more effective than the other IL-6 family cytokines in causing ERK, JNK, and STAT3 phosphorylation in HASM cells (11,28). Differences in ERK and JNK activation do not appear to contribute to differences in the ability of OSM vs. other IL-6 family cytokines to induce VEGF release, since neither the MEK inhibitor U0126, which inhibits ERK phosphorylation and subsequent activation, nor the JNK inhibitor SP-600125 had any effect on OSM-induced VEGF release.…”

Oncostatin M causes eotaxin-1 release from airway smooth muscle: Synergy with IL-4 and IL-13

“…Luciferase and b-gal were assayed as previously described. 17,28 Western blotting Confluent cells were treated with 20 ng/mL cytokines for up to 4 hours and then lysed, and solubilized proteins (30 mg per lane) were separated by means of SDS-PAGE and transferred to a BioTrace polyvinylidene difluoride membrane, as previously described. 11 The membranes were blocked and then probed with rabbit antiphosphoTyr705-STAT-3, anti-phospho-p44/p42, anti-phospho-JNK, or anti-phospho-p38 (Cell Signaling, Inc, Woburn, Mass) overnight at 4°C.…”