PKCbetaII augments NF-kappaB-dependent transcription at the CCL11 promoter via p300/CBP-associated factor recruitment and histone H4 acetylation.

The transcription factor NF-kappaB plays a pivotal role in regulating inflammatory gene expression. Its effects are optimized by various coactivators, including histone acetyltransferases (HATs) such as CREB-binding protein/p300 and p300/CBP-associated factor (p/CAF). The molecular mechanisms regulating cofactor recruitment are poorly understood. In this study, we describe a novel role for protein kinase C (PKC) betaIotaIota in augmenting NF-kappaB-mediated TNF-alpha-induced transcription of the target gene CCL11 in human airway smooth muscle cells by phosphorylating the HAT p/CAF. Studies using reporters, overexpression strategies, kinase-dead and HAT-defective mutants, and chromatin immunoprecipitation showed that PKCbetaII activation was not involved in NF-kappaB translocation, but facilitated NF-kappaB-mediated CCL11 transcription by colocalizing with and phosphorylating p/CAF, and thereby acetylating histone H4 and promoting p65 association with the CCL11 promoter. The effect was dependent on p/CAF's HAT activity. Furthermore, mouse embryonic fibroblasts from PKCbeta knockout mice showed markedly reduced TNF-alpha-induced CCL11 expression and NF-kappaB reporter activity that was restored on PKCbetaII overexpression, suggesting a critical role for this pathway. These data suggest a novel important biological role for PKCbetaIotaIota in NF-kappaB-mediated CCL11 transcription by p/CAF activation and histone H4 acetylation.

Cytokines upregulate vascular endothelial growth factor secretion by human airway smooth muscle cells: Role of endogenous prostanoids.

Here, we report that vascular endothelial growth factor (VEGF)-A secretion by human airway smooth muscle cells was increased by interleukin 1 beta (IL-1beta) and transforming growth factor beta (TGFbeta). IL-1beta and TGFbeta induced cyclo-oxygenase (COX)-2 protein and increased prostaglandin E(2) (PGE(2)). Both IL-1beta and TGFbeta increased VEGF-A(165) mRNA and VEGF promoter luciferase construct activity, in addition VEGF-A protein was inhibited by actinomycin D suggesting transcriptional regulation. The COX inhibitors indomethacin and NS398 inhibited IL-1beta but not TGFbeta mediated VEGF-A production. Furthermore, the effect of the COX inhibitors was overcome by adding exogenous PGE(2). In conclusion, IL-1beta increases VEGF-A secretion by COX-2 derived PGE(2) production whereas TGFbeta uses COX-independent pathways.

Cyclooxygenase (COX) inhibitors induce apoptosis in non-small cell lung cancer through cyclooxygenase independent pathways.

Cyclooxygenase (COX) inhibitors are chemopreventive in many tumours but the role of COX inhibition in their effects is contentious. Here we determined if COX inhibitors influenced apoptosis in two non-small cell lung cancer cells one which over expresses COX-2 (MOR-P) and one which expresses neither isoform (H-460). NS398, a selective COX inhibitor, and indomethacin, a non-selective COX inhibitor, were cytotoxic in both cell lines, independently of their COX-2 expression. Furthermore, the cytotoxic concentrations were far greater than the concentrations required to inhibit COX. As indomethacin was more effective we used it in mechanistic studies. Indomethacin induced apoptotic cell death assessed as cytochrome c and apoptotic inducing factor (AIF) release, caspase activation, PARP, lamin B and gelsolin cleavage, chromatin condensation and nuclear fragmentation. The pan-caspase inhibitor, z-VAD, attenuated cell death, and blocked caspase activation, PARP cleavage and nuclear fragmentation without preventing cytochrome c release, suggesting that cytochrome c release is upstream of caspase activation. These observations suggest that COX inhibitors induce apoptosis in non-small lung cancer cells through cytochrome c and AIF release, and subsequent caspase activation, independently of COX-2 expression and prostaglandin production.

Transcriptional regulation of monocyte chemotactic protein-1 release by endothelin-1 in human airway smooth muscle cells involves NF-kappaB and AP-1.

BACKGROUND AND PURPOSE: Endothelin-1 (ET-1) is implicated in airway inflammation in asthma, but the mechanisms of its effects are poorly understood. We studied the effect of ET-1 on expression of the chemokine, monocyte chemotactic protein-1 (MCP-1), in primary cultures of human airway smooth muscle cells. EXPERIMENTAL APPROACH: MCP-1 release was measured by elisa. Pharmacological antagonists/inhibitors, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting were used to study ET receptors and kinase cascades. Transcriptional regulation was studied by real-time RT-PCR, transient transfection studies and chromatin immunoprecipitation assay. Major findings were confirmed in cells from three donors and mechanistic studies in cells from one donor. KEY RESULTS: ET-1 increased MCP-1 release through an ET(A) and ET(B) receptor-dependent mechanism. ET-1 increased MCP-1 mRNA levels but not mRNA stability suggesting it was acting transcriptionally. ET-1 increased the activity of an MCP-1 promoter-reporter construct. Serial deletions of the MCP-1 promoter mapped ET-1 effects to a region between -213 and -128 base pairs upstream of the translation start codon, containing consensus sequences for activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB). ET-1 promoted binding of AP-1 c-Jun subunit and NF-kappaB p65 subunit to the MCP-1 promoter. Blocking the inhibitor of kappaB kinase-2 with 2-[(aminocarbonyl)amino]-5-[4-fluorophenyl]-3-thiophenecarboxamide (TPCA-1) decreased ET-1-stimulated MCP-1 production. p38 and p44/p42 mitogen-activated protein kinases were involved in upstream signalling. CONCLUSIONS AND IMPLICATIONS: ET-1 regulated MCP-1 transcriptionally, via NF-kappaB and AP-1. The upstream signalling involved ET(A), ET(B) receptors, p38 and p44/p42 mitogen-activated protein kinases. These may be targets for novel asthma therapies.

Vascular endothelial growth factor induction by prostaglandin E2 in human airway smooth muscle cells is mediated by E prostanoid EP2/EP4 receptors and SP-1 transcription factor binding sites.

Prostaglandin E2 (PGE2) can increase endothelial vascular endogrowth factor A (VEGF-A) production but the mechanisms involved are unclear. Here we characterized the transcriptional mechanisms involved in human airway smooth muscle cells (HASMC). PGE2 increased VEGF-A mRNA and protein but not mRNA stability. PGE2 stimulated the activity of a transiently transfected 2068-bp (-2018 to +50) VEGF-A promoter-driven luciferase construct. Functional 5' deletional analysis mapped the PGE2 response element to the 135-bp sequence (-85/+50) of the human VEGF-A promoter. PGE2-induced luciferase activity was reduced in cells transfected with a 135-bp VEGF promoter fragment containing mutated Sp-1 binding sites but not in cells transfected with a construct containing mutated EGR-1 binding sites. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay confirmed binding of Sp-1 to the VEGF promoter. PGE2 increased phosphorylation of Sp-1 and luciferase activity of a transfected Sp-1 reporter construct. PGE receptor agonists EP2 (ONO-AE1 259) and EP4 (ONO-AE1 329) mimicked the effect of PGE2, and reverse transcription-PCR, Western blotting, and flow cytometry confirmed the presence of EP2 and EP4 receptors. VEGF protein release and Sp-1 reporter activity were increased by forskolin and isoproterenol, which increase cytosolic cAMP, and the cAMP analogue, 8-bromoadenosine-3',5'-cyclophosphoric acid. These studies suggest that PGE2 increases VEGF transcriptionally and involves the Sp-1 binding site via a cAMP-dependent mechanism involving EP2 and EP4 receptors.

Transcriptional regulation of interleukin (IL)-8 by bradykinin in human airway smooth muscle cells involves prostanoid-dependent activation of AP-1 and nuclear factor (NF)-IL-6 and prostanoid-independent activation of NF-kappaB.

Bradykinin (BK) is a potent neutrophil chemotractant, proinflammatory mediator, and angiogenic factor, which acts through G protein-coupled receptors (GPCRs). Here we studied the mechanisms involved in IL-8 generation by BK in human airway smooth muscle cells focusing on the transcription factors involved and role of endogenous prostanoids in transcription factor activation. Transfection experiments with wild-type IL-8 promoter constructs or constructs with NF-kappaB, AP-1, and NF-IL-6 binding site mutations suggested that all three transcription factors were necessary for optimal IL-8 expression. BK increased NF-kappaB, AP-1, and NF-IL-6 binding to the IL-8 promoter by electrophoretic mobility shift assay. NF-kappaB, the most important transcription factor in the current study, was translocated to the nucleus after BK stimulation. Indomethacin, a cyclooxygenase inhibitor, partially inhibited IL-8 release and the promoter binding of AP-1 and NF-IL-6, but not NF-kappaB. Furthermore, exogenous prostaglandin E2 stimulated AP-1 and NF-IL-6 binding to the IL-8 promoter. The anti-inflammatory glucocorticoid dexamethasone inhibited NF-kappaB translocation and the promoter binding of NF-kappaB, AP-1, and NF-IL-6. These results are the first to delineate the transcription factors involved in BK induced IL-8 release. Transcriptional activation of the IL-8 promoter by BK involves the prostanoid-independent activation of NF-kappaB, and prostanoid-dependent activation of AP-1 and NF-IL-6 plays a key role in augmenting the response. Endogenous prostanoid generation in response to GPCR ligands such as BK may be an important mechanism whereby GPCRs signal to the nucleus to maximize the transcription of inflammatory response genes.

Cyclooxygenase-2 induction by bradykinin in human pulmonary artery smooth muscle cells is mediated by the cyclic AMP response element through a novel autocrine loop involving endogenous prostaglandin E2, E-prostanoid 2 (EP2), and EP4 receptors.

Bradykinin (BK) is an important mediator in several inflammatory and vascular diseases that acts in part via induction of cyclooxygenase-2 (COX-2). The mechanisms involved in BK-mediated COX-2 induction are unclear. Here we characterized the transcriptional mechanisms involved in human pulmonary artery smooth muscle cells. BK stimulated the activity of a transiently transfected 966-bp (-917 to + 49) COX-2 promoter luciferase reporter construct. There was no reduction in BK-induced luciferase activity in cells transfected with COX-2 promoter constructs of 674, 407, 239, and 135 bp or constructs with mutated CCAAT/enhancer-binding protein- or NF-kappaB-binding sites. In contrast luciferase activity was reduced in cells transfected with a 407-bp COX-2 promoter fragment containing a mutated cAMP response element (CRE)-binding site, suggesting that the CRE binding site is critical. Electrophoretic mobility shift assays using oligonucleotides specific for the CRE-binding region of the COX-2 promoter and consensus oligonucleotides showed strong specific binding. Furthermore BK increased consensus cAMP-responsive luciferase reporter (p6CRE/luc)-mediated luciferase expression. CRE activation occurred by BK inducing cytosolic phospholipase A2-mediated arachidonic acid release and rapid prostaglandin E2 (PGE2) production, thereby increasing cAMP. Indomethacin inhibited BK-induced PGE2 production, cAMP accumulation, and CRE/luc reporter and COX-2 promoter luciferase activity. Exogenous PGE2 and EP2 (ONO-AE1 259) and EP4 (ONO-AE1 329) PGE2 receptor agonists mimicked the effect of BK. Collectively these studies indicate that COX-2 induction by BK in human pulmonary artery smooth muscle cells is mediated by the CRE through a novel autocrine loop involving endogenous PGE2.