Activation of AP-1 contributes to the beta-adrenoceptor-mediated myocardial induction of interleukin-6

The induction of proinflammatory cytokines in stressed myocardium is considered an innate immune response, but the role of beta-adrenergic signaling in this proinflammatory response and the mechanisms of cardioprotection by beta-blockers are not fully understood. In the present study, we analyzed interleukin-6 (IL-6) formation and promoter activation in beta-adrenoceptor-stimulated neonatal rat cardiomyocytes, in transgenic mice with cardiac overexpression of beta1-adrenoceptors, and in failing human myocardium. IL-6 formation and release in cultured cardiomyocytes under beta-adrenoceptor stimulation requires the activation of activating protein-1 (AP-1) binding sites and of cAMP response elements (CRE) in the IL-6 promoter, but this release (140 +/- 6 pg/mL medium under 10(-6) M isoproterenol vs. 81 +/- 3 pg/mL unstimulated, P < 0.05) is moderate compared with that under inflammatory stimulation (855 +/- 44 pg/mL, endotoxin 0.1microg/mL). Similarly, IL-6 is induced together with CRE- and AP-1 activation in the left ventricle (LV) of beta1-transgenic mice before the onset of failure. However, we observed IL-6 induction with activation of NF-kappaB in addition to CRE and AP-1 in beta1-transgenic mice at the age of 22 weeks and in explanted human LV after full development of failure. Treatment with beta-blockers lowered myocardial IL-6 as well as AP-1, NF-kappaB, and CRE activation. Therefore, the activation of AP-1 and CRE is part of beta-adrenergic signal transduction for IL-6 induction in nonfailing and failing cardiomyocytes, whereas NF-kappaB activation contributes only in overloaded failing myocardium.     

The anti-inflammatory effects of heat shock protein 72 involve inhibition of high-mobility-group box 1 release and proinflammatory function in macrophages

High-mobility-group box 1 (HMGB1), a nuclear protein, has recently been identified as an important mediator of local and systemic inflammatory diseases when released into the extracellular milieu. Anti-inflammatory regulation by the stress response is an effective autoprotective mechanism when the host encounters harmful stimuli, but the mechanism of action remains incompletely delineated. In this study, we demonstrate that increases in levels of a major stress-inducible protein, heat shock protein 72 (Hsp72) by gene transfection attenuated LPS- or TNF-alpha-induced HMGB1 cytoplasmic translocation and release. The mechanisms involved inhibition of the chromosome region maintenance 1 (CRM1)-dependent nuclear export pathway. Overexpression of Hsp72 inhibited CRM1 translocation and interaction between HMGB1 and CRM1 in macrophages post-LPS and TNF-alpha treatment. In addition, overexpression of Hsp72 strongly inhibited HMGB1-induced cytokine (TNF-alpha, IL-1beta) expression and release, which correlated closely with: 1) inhibition of the MAP kinases (p38, JNK, and ERK); and 2) inhibition of the NF-kappaB pathway. Taken together, these experiments suggest that the anti-inflammatory activity of Hsp72 is achieved by interfering with both the release and proinflammatory function of HMGB1. Our experimental data provide important insights into the anti-inflammatory mechanisms of heat shock protein protection.     

Phosphatidylinositol 3-kinase/Akt signaling mediates interleukin-32alpha induction in human pancreatic periacinar myofibroblasts

Interleukin (IL)-32 is a recently described proinflammatory cytokine, characterized by the induction of nuclear factor (NF)-kappaB activation. We studied IL-32alpha expression in human pancreatic periacinar myofibroblasts, which play important roles in the regulation of extracellular matrix metabolism and inflammatory responses in the pancreas. IL-32alpha protein expression was evaluated by Western blot analyses, and IL-32alpha mRNA expression was analyzed by Northern blot and real-time PCR analyses. IL-32alpha mRNA was weakly expressed without a stimulus, and its expression was markedly enhanced by IL-1beta, IFN-gamma, and TNF-alpha. IL-1beta, IFN-gamma, and TNF-alpha enhanced intracellular accumulation of IL-32alpha protein, but IL-32alpha was not detected in supernatants. Each cytokine dose and time dependently induced IL-32alpha mRNA expression. An inhibitor of phosphatidylinositol 3-kinase (LY294002) significantly suppressed IL-1beta-, IFN-gamma-, and TNF-alpha-induced IL-32alpha mRNA expression, although MAPK inhibitors had no effect. Akt activation in response to these cytokines was confirmed by Western blot. Furthermore, LY294002 suppressed both IL-1beta- and TNF-alpha-induced NF-kappaB activation and IL-1beta-, TNF-alpha-, and IFN-gamma-induced activated protein-1 (AP-1) activation. Blockade of NF-kappaB and AP-1 activation by an adenovirus expressing a stable mutant form of IkappaBalpha and a dominant negative mutant of c-Jun markedly suppressed IL-1beta-, IFN-gamma-, and/or TNF-alpha-induced IL-32alpha mRNA expression. Human pancreatic periacinar myofibroblasts expressed IL-32alpha in response to IL-1beta, TNF-alpha, and IFN-gamma. IL-32alpha mRNA expression is dependent on interactions between the phosphatidylinositol 3-kinase/Akt-pathway and the NF-kappaB/AP-1 system.     

Suppressive effect of dexamethasone on TIMP-1 production involves murine osteoblastic MC3T3-E1 cell apoptosis

High dose glucocorticoid (GC) treatment induces osteoporosis partly via increasing osteoblast apoptosis. However, the mechanism of GC-induced apoptosis has not been fully elucidated. Osteoblast-derived tissue inhibitor of metalloproteinase-1 (TIMP-1) was recently reported to be involved in bone metabolism. Our previous study demonstrated that TIMP-1 suppressed apoptosis of the mouse bone marrow stromal cell line MBA-1 (pre-osteoblast) induced by serum deprivation. Therefore, we tested the effect of the GC dexamethasone (Dex) on TIMP-1 production in murine osteoblastic MC3T3-E1 cells and further determined whether this action is associated with Dex-induced osteoblast apoptosis. Dex decreased TIMP-1 production in MC3T3-E1 cells, and this effect was blocked by the glucocorticoid receptor (GR) antagonists, RU486 and RU40555. Recombinant TIMP-1 protein reduced caspase-3 activation and apoptosis induced by Dex in MC3T3-E1 cells. In addition, the pro-apoptotic effect of the Dex was augmented by suppression of TIMP-1 with siRNA. Furthermore, mutant TIMP-1, which has no inhibitory effects on MMPs, yet protects MC3T3-E1 cells against Dex-induced apoptosis. Our study demonstrates that Dex suppresses TIMP-1 production in osteoblasts through GR, and this effect is associated with its induction of osteoblast apoptosis. The anti-apoptotic action of TIMP-1 is independent of its inhibitory effects on MMPs activities. The decrease in TIMP-1 production caused by Dex may contribute to the mechanisms of Dex-induced bone loss.     

Astaxanthin inhibits nitric oxide production and inflammatory gene expression by suppressing I(kappa)B kinase-dependent NF-kappaB activation

Astaxanthin, a carotenoid without vitamin A activity, has shown anti-oxidant and anti-inflammatory activities; however, its molecular action and mechanism have not been elucidated. We examined in vitro and in vivo regulatory function of astaxanthin on production of nitric oxide (NO) and prostaglandin E2 (PGE2) as well as expression of inducible NO synthase (iNOS), cyclooxygenase-2, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta). Astaxanthin inhibited the expression or formation production of these proinflammatory mediators and cytokines in both lipopolysaccharide (LPS)-stimulated RAW264.7 cells and primary macrophages. Astaxanthin also suppressed the serum levels of NO, PGE2, TNF-alpha, and IL-1beta in LPS-administrated mice, and inhibited NF-kappaB activation as well as iNOS promoter activity in RAW264.7 cells stimulated with LPS. This compound directly inhibited the intracellular accumulation of reactive oxygen species in LPS-stimulated RAW264.7 cells as well as H2O2-induced NF-kappaB activation and iNOS expression. Moreover, astaxanthin blocked nuclear translocation of NF-kappaB p65 subunit and I(kappa)B(alpha) degradation, which correlated with its inhibitory effect on I(kappa)B kinase (IKK) activity. These results suggest that astaxanthin, probably due to its antioxidant activity, inhibits the production of inflammatory mediators by blocking NF-kappaB activation and as a consequent suppression of IKK activity and I(kappa)B-alpha degradation.