Oxidative stress as a result of cigarette smoking is an important etiologic factor in the pathogenesis of chronic obstructive pulmonary disease (COPD), a chronic steroid-insensitive inflammatory disease of the airways. Histone deacetylase-2 (HDAC2), a critical component of the corticosteroid anti-inflammatory action, is impaired in lungs of patients with COPD and correlates with disease severity. We demonstrate here that curcumin (diferuloylmethane), a dietary polyphenol, at nanomolar concentrations specifically restores cigarette smoke extract (CSE)-or oxidative stress-impaired HDAC2 activity and corticosteroid efficacy in vitro with an EC 50 of approximately 30 nM and 200 nM, respectively. CSE caused a reduction in HDAC2 protein expression that was restored by curcumin. This decrease in HDAC2 protein expression was reversed by curcumin even in the presence of cycloheximide, a protein synthesis inhibitor. The proteasomal inhibitor, MG132, also blocked CSE-induced HDAC2 degradation, increasing the levels of ubiquitinated HDAC2. Biochemical and gene chip analysis indicated that curcumin at concentrations up to 1 mM propagates its effect via antioxidant-independent mechanisms associated with the phosphorylation-ubiquitin-proteasome pathway. Thus curcumin acts at a post-translational level by maintaining both HDAC2 activity and expression, thereby reversing steroid insensitivity induced by either CSE or oxidative stress in monocytes. Curcumin may therefore have potential to reverse steroid resistance, which is common in patients with COPD and asthma.

COPD is a disease of innate immunity and bacterial infections are a dominant cause of exacerbations in the later stages resulting in poor health and high mortality. The pathogen-associated molecular pattern (PAMP) lipopolysaccharide (LPS) is sensed by immune cells through activation of the toll-like receptor 4 (TLR4). This leads to the activation of NADPH oxidase (NOX) and NF-κB which together drive COPD inflammation. In this study we show in human PBMCs that LPS stimulated proinflammatory cytokine release (CXCL8 and IL6) was inhibited by approximately 50% by the broad specificity phosphatidylinositol 3-kinase (PI3K) inhibitor, wortmannin. Our results also demonstrate that activation of PI3K following LPS stimulation is mediated by a NOX4 dependent mechanism releasing endogenous H2O2, as the NOX4 inhibitor apocynin blocked LPS induced AKT phosphorylation. Moreover, LPS-induced PI3K activation was inhibited by the anti-oxidant N-acetylcysteine in a concentration dependent manner (IC50 ~100 μM). In addition, our data demonstrated that inhibition of small G proteins, by pre-treatment with pertussis toxin, inhibited LPS-induced AKT phosphorylation. Furthermore, the G-protein inhibitors pertussis toxin and mastoparan both inhibited LPS-induced CXCL8 and IL-6 release by approximately 50%. Together, these data indicate there is a mechanism in human PBMCs where TLR4 activation by LPS leads to ROS generation through NOX4 and activation of the PI3K pathway. This effect is apparently mediated through small G proteins facilitating the release of pro-inflammatory cytokines.

cAMP-elevating drugs are thought to mediate their biological effects by activating the cAMP/cAMP-dependent protein kinase (PKA) cascade. However, this hypothesis is difficult to confirm due to a lack of selective inhibitors. Here, we have probed the role of PKA in mediating inhibitory effects of several cAMP-elevating drugs in BEAS-2B epithelial cells using an adenovirus vector encoding a PKA inhibitor protein (PKI␣) and have compared it to H-89, a commonly used small molecule PKA inhibitor. Initial studies established efficient gene transfer and confirmed functionality of PKI␣ 48 h after virus infection. All cAMP-elevating drugs tested promoted the phosphorylation of cAMP response element-binding protein (CREB), activated a cAMP response element (CRE)-driven luciferase reporter gene, and suppressed both granulocyte/ macrophage colony-stimulating factor (GM-CSF) generation and [ 3 H]arachidonic acid (AA) release in response to interleukin-1␤ and monocyte chemotactic protein (MCP)-1, respectively. These effects were abolished by PKI␣. In contrast, H-89 behaved unpredictably under the same conditions. Thus, although CREB phosphorylation evoked by a range of cAMP-elevating drugs was abolished by H-89, neither activation of the CRE-dependent luciferase reporter gene construct nor the inhibition of GM-CSF generation was inhibited. Paradoxically, H-89 antagonized MCP-1-induced [ 3 H]AA release and enhanced the inhibitory effect of submaximal concentrations of rolipram and 8-bromo-cAMP. We suggest that expression of PKI␣ in susceptible cells provides a simple and unambiguous way to assess the role of PKA in cAMP signaling and to probe the mechanism of action of other drugs and cAMP-dependent responses where the participation of PKA is equivocal. Furthermore, these data suggest that H-89 is not a selective inhibitor of PKA and should be avoided.Through highly coordinated changes in the rate of synthesis and degradation, cAMP mediates the effect of a large number of hormones, autacoids, and neurotransmitters. Current dogma holds that agonism of Gs-coupled receptors augments the basal activity of one or more isoforms of adenylyl cyclase. The cAMP signal then is propagated and amplified through the activation of PKA, ultimately to effect a change in cell function (see Beavo and Brunton, 2002). In the inactive state, PKA is a tetramer composed of two catalytic and two regulatory subunits (Francis and Corbin, 1999). cAMP, when elevated, binds to the regulatory subunits, resulting in the dissociation of the inactive holoenzyme and the release of

1 The prostanoid receptor(s) that mediates inhibition of bacterial lipopolysaccharide (LPS)-induced tumour necrosis factor-a (TNFa) generation from human peripheral blood monocytes was classi®ed by use of naturally occurring and synthetic prostanoid agonists and antagonists. 2 In human monocytes that were adherent to plastic, neither prostaglandin D 2 (PGD 2 ), prostaglandin E 2 (PGE 2 ), prostaglandin F 2a (PGF 2a ) nor the stable prostacyclin and thromboxane mimetics, cicaprost and U-46619, respectively, promoted the elaboration of TNFa-like immunoreactivity, as assessed with a speci®c ELISA, indicating the absence of excitatory prostanoid receptors on these cells. 3 Exposure of human monocytes to LPS (3 ng ml 71 , * EC 84 ) resulted in a time-dependent elaboration of TNFa which was suppressed in cells pretreated with prostaglandin E 1 (PGE 1 ), PGE 2 and cicaprost. This e ect was concentration-dependent with mean pIC 50 values of 7.14, 7.34 and 8.00 for PGE 1 , PGE 2 and cicaprost, respectively. PGD 2 , PGF 2a and U-46619 failed to inhibit the generation of TNFa at concentrations up to 10 mM. 4 With respect to PGE 2 , the EP-receptor agonists, 16,16-dimethyl PGE 2 (non-selective), misoprostol (EP 2 /EP 3 -selective), 11-deoxy PGE 1 (EP 2 -selective) and butaprost (EP 2 -selective) were essentially full agonists as inhibitors of LPS-induced TNFa generation with mean pIC 50 values of 6.21, 6.02, 5.67 and 5.59, respectively. In contrast to the results obtained with butaprost and 11-deoxy PGE 1 , another EP 2 -selective agonist, AH 13205, inhibited TNFa generation by only 21% at the highest concentration (10 mM) examined. EP-receptor agonists which have selectivity for the EP 1 -(17-phenyl-o-trinor PGE 2 ) and EP 3 -receptor (MB 28,767, sulprostone) were inactive or only weakly active as inhibitors of TNFa generation. 5 Pretreatment of human monocytes with the TP/EP 4 -receptor antagonist, AH 23848B, at 10, 30 and 100 mM suppressed LPS-induced TNFa generation by 10%, 28% and 77%, respectively, but failed to shift signi®cantly the location of the PGE 2 concentration-response curves. 6 Given that AH 13205 was a poor inhibitor of TNFa generation, studies were performed to determine if it was a partial agonist and whether it could antagonize the inhibitory e ect of PGE 2 . Pretreatment of human monocytes with 10 and 30 mM AH 13205 inhibited the generation of TNFa by 31% and 53%, respectively, but failed to shift signi®cantly the location of the PGE 2 concentration-response curves at either concentration examined. 7 Since PGD 2 and 17-phenyl-o-trinor PGE 2 (EP 1 -agonist) did not suppress TNFa generation, the EP 1 / EP 2 /DP-receptor antagonist, AH 6809, was employed to assess if EP 2 -receptors mediated the inhibitory e ect of PGE 2 . Pretreatment of human monocytes with 10 mM AH 6809 did not a ect LPS-induced TNFa generation but produced a parallel 3.5 fold rightwards shift of the PGE 2 concentration-response curve. 8 Collectively, these data suggest that human peripheral blood monocytes express at least two distin...