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Background and purpose Haem oxygenase-1 (HO-1) is induced by thiazolidinediones including rosiglitazone and exerts anti-inflammatory effects in various models. However, the molecular mechanisms underlying rosiglitazone-induced HO-1 expression remain largely unknown in human pulmonary alveolar epithelial cells (HPAEpiCs). Experimental approach HO-1 expression was determined by real time-PCR, Western blotting and promoter reporter analyses. Signalling pathways were investigated using pharmacological inhibitors or specific siRNAs. Interactions between nuclear factor erythroid-2-related factor (Nrf2) and antioxidant response elements (ARE) binding site of the HO-1 promoter were investigated with chromatin immunoprecipitation assays. Key results Up-regulation of HO-1 in HPAEpiCs or in mice by rosiglitazone blunted ICAM-1 expression and monocyte adhesion to HPAEpiCs challenged with LPS. Rosiglitazone-induced HO-1 expression was significantly attenuated by NADPH oxidase (NOX) inhibitors (apocynin and diphenyleneiodonium) or ROS scavenger (N-acetyl cysteine). The involvement of NOX activity and ROS generation in rosiglitazone-induced HO-1 expression was confirmed by transfection with p47phox or NOX2 siRNA. Moreover, pretreatment with the inhibitors of c-Src (c-Srci II), proline-rich tyrosine kinase 2 (Pyk2) (PF431396), Akt (Akti VIII) or PPARγ (GW9662) and transfection with siRNA of c-Src, Pyk2, Akt or PPARγ abolished the rosiglitazone-induced HO-1 expression in HPAEpiCs. Subsequently, Nrf2 was activated by phosphorylation of c-Src, Pyk2 and Akt, which turned on transcription of HO-1 gene by binding to AREs binding site and enhancing ARE promoter activity. Conclusions and implications Rosiglitazone induces HO-1 expression via either NOX/ROS/c-Src/Pyk2/Akt-dependent Nrf2 activation or PPARγ in HPAEpiCs and suppresses LPS-mediated inflammatory responses, suggesting that PPARγ agonists may be useful for protection against pulmonary inflammation.

Neuroinflammation is a landmark of neuroinflammatory and neurodegenerative diseases. Matrix metalloproteinase (MMP)-9, one member of MMPs, has been shown to contribute to the pathology of these brain diseases. Several experimental models have demonstrated that lipopolysaccharide (LPS) exerts a pathological role through Toll-like receptors (TLRs) in neuroinflammation and neurodegeneration. However, the mechanisms underlying LPS-induced MMP-9 expression in rat brain astrocytes (RBA-1) are not completely understood. Here, we applied pharmacological inhibitors and siRNA transfection to assess the levels of MMP-9 protein, mRNA, and promoter activity, as well as protein kinase phosphorylation in RBA-1 cells triggered by LPS. We found that LPS-induced expression of pro-form MMP-9 and cell migration were mediated through TLR4, proto-oncogene tyrosine-protein kinase (c-Src), proline-rich tyrosine kinase 2 (Pyk2), platelet-derived growth factor receptor (PDGFR), phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt), p38 mitogen-activated protein kinase (MAPK), and Jun amino-terminal kinase (JNK)1/2 signaling molecules in RBA-1 cells. In addition, LPS-stimulated binding of c-Jun to the MMP-9 promoter was confirmed by chromatin immunoprecipitation (ChIP) assay, which was blocked by pretreatment with c-Src inhibitor II, PF431396, AG1296, LY294002, Akt inhibitor VIII, p38 MAP kinase inhibitor VIII, SP600125, and tanshinone IIA. These results suggest that in RBA-1 cells, LPS activates a TLR4/c-Src/Pyk2/PDGFR/PI3K/Akt/p38 MAPK and JNK1/2 pathway, which in turn triggers activator protein 1 (AP-1) activation and ultimately induces MMP-9 expression and cell migration.

Background: Mevastatin (MVS), a 3-hydroxy-3-methylglutaryl coenzyme, a reductase (HMG-CoA) inhibitor, has anti-inflammatory effects potentially via up-regulation of heme oxygenase-1 (HO-1). However, the mechanisms underlying MVS-induced HO-1 expression remain largely unknown in human pulmonary alveolar epithelial cells (HPAEpiCs). Methods: HO-1 and intercellular adhesion molecule (ICAM)-1 expression were determined using real-time PCR, Western blotting, and promoter reporter analyses. The signaling components were investigated using pharmacological inhibitors or specific small interfering RNA (siRNA)s. Interaction between Nrf2 and the antioxidant response element (ARE) binding site for the HO-1 promoter was determined by chromatin immunoprecipitation (ChIP) assay. Results: Upregulation of HO-1 by MVS attenuated the tumor necrosis factor (TNF)-&alpha, stimulated ICAM-1 expression associated with THP-1 adhesion to HPAEpiCs. These inhibitory effects of HO-1 were reversed by tin protoporphyrin (SnPP)IX or by transfection with HO-1 siRNA. MVS-induced HO-1 expression was mediated via NADPH oxidase (Nox)-derived reactive oxygen species (ROS) generation. Activation of Nox2/ROS further stimulated the phosphorylation of p47phox, proto-oncogene tyrosine-protein kinase (c-Src), platelet-derived growth factor receptor (PDFGR)&alpha, protein kinase B (Akt), and Nrf2, which were inhibited by siRNAs. Pretreatment with pharmacological inhibitors, including diphenyleneiodonium (DPI), apocynin (APO), N-acetyl-L-cysteine (NAC), PP1, AG1296, or LY294002, reduced the MVS-activated Nrf2 nuclear-translocation binding to the ARE on the HO-1 promoter. Conclusions: MVS-induced HO-1 is, at least in part, mediated through a p47phox/Nox2/ROS-dependent activation of c-Src/PDGFR&alpha, /PI3K/Akt-regulated Nrf2/ARE axis and suppresses the TNF-&alpha, mediated inflammatory responses in HPAEpiCs.

Lysophosphatidylcholine (LysoPC) has been shown to induce the expression of inflammatory proteins, including cyclooxygenase-2 (COX-2) and interleukin-6 (IL-6), associated with cardiac fibrosis. Here, we demonstrated that LysoPC-induced COX-2 and IL-6 expression was inhibited by silencing NADPH oxidase 1, 2, 4, 5; p65; and FoxO1 in human cardiac fibroblasts (HCFs). LysoPC-induced IL-6 expression was attenuated by a COX-2 inhibitor. LysoPC-induced responses were mediated via the NADPH oxidase-derived reactive oxygen species-dependent JNK1/2 phosphorylation pathway, leading to NF-κB and FoxO1 activation. In addition, we demonstrated that both FoxO1 and p65 regulated COX-2 promoter activity stimulated by LysoPC. Overexpression of wild-type FoxO1 and S256D FoxO1 enhanced COX-2 promoter activity and protein expression in HCFs. These results were confirmed by ex vivo studies, where LysoPC-induced COX-2 and IL-6 expression was attenuated by the inhibitors of NADPH oxidase, NF-κB, and FoxO1. Our findings demonstrate that LysoPC-induced COX-2 expression is mediated via NADPH oxidase-derived reactive oxygen species generation linked to the JNK1/2-dependent pathway leading to FoxO1 and NF-κB activation in HCFs. LysoPC-induced COX-2-dependent IL-6 expression provided novel insights into the therapeutic targets of the cardiac fibrotic responses.

Bradykinin (BK) is a proinflammatory mediator and elevated in several brain injury and inflammatory diseases. The deleterious effects of BK on brain astrocytes may aggravate brain inflammation mediated through the upregulation of cytosolic phospholipase A2 (cPLA2)/cyclooxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) production. However, the signaling mechanisms underlying BK-induced cPLA2 expression in brain astrocytes remain unclear. Herein, we investigated the effects of activation of cPLA2/COX-2 system on BK-induced cPLA2 upregulation in rat brain astrocytes (RBA-1). The data obtained with Western blotting, RT-PCR, and immunofluorescent staining analyses showed that BK-induced de novo cPLA2 expression was mediated through activation of cPLA2/COX-2 system. Upregulation of native cPLA2/COX-2 system by BK through activation of PKCδ, c-Src, MAPKs (ERK1/2 and JNK1/2) cascades led to PGE2 biosynthesis and release. Subsequently, the released PGE2 induced cPLA2 expression via the same signaling pathways (PKCδ, c-Src, ERK1/2, and JNK1/2) and then activated the cyclic AMP response element-binding protein (CREB) via B2 BK receptor-mediated cPLA2/COX-2 system-derived PGE2/EP-dependent manner. Finally, upregulation of cPLA2 by BK may promote more PGE2 production. These results demonstrated that in RBA-1, activation of CREB by PGE2/EP-mediated PKCδ/c-Src/MAPK cascades is essential for BK-induced de novo cPLA2 protein. More importantly, upregulation of cPLA2 by BK through native cPLA2/COX-2 system may be a positive feedback mechanism that enhances prolonged brain inflammatory responses. Understanding the mechanisms of cPLA2/COX-2 system upregulated by BK on brain astrocytes may provide rational therapeutic interventions for brain injury and inflammatory diseases.

The inflammation-dependent adhesion molecule expressions are characterized in cardiovascular diseases and myocardial tissue infiltrations. Several pro-inflammatory cytokines are elevated in the acute myocardial injury and infarction. Tumor necrosis factor-α (TNF-α), a pro-inflammatory cytokine, is raised in the injury tissues and inflammatory regions and involved in the pathogenesis of cardiac injury, inflammation, and apoptosis. In fibroblasts, TNF-α-triggered expression of vascular cell adhesion molecule (VCAM)-1 aggravated the heart inflammation. However, the mechanisms underlying TNF-α-mediated VCAM-1 expression in cardiac fibroblasts remain unclear. Here, the primary cultured human cardiac fibroblasts (HCFs) were used to investigate the effects of TNF-α on VCAM-1 expression. The molecular evidence, including protein, mRNA, and promoter analyses, indicated that TNF-α-induced VCAM-1 gene expression is mediated through the TNFR-dependent manner. Activation of TNF-α/TNFR system triggered PKCα-dependent NADPH oxidase (Nox)/reactive oxygen species (ROS) signal linking to MAPK cascades, and then led to activation of the transcription factor, AP-1. Moreover, the results of mRNA and promoter assay demonstrated that c-Jun/AP-1 phosphorylated by TNF-α turns on VCAM-1 gene expression. Subsequently, up-regulated VCAM-1 on the cell surface of TNF-α-challenged HCFs increased the number of monocytes adhering to these cells. These results indicated that in HCFs, activation of AP-1 by PKCα-dependent Nox/ROS/MAPKs cascades is required for TNF-α-induced VCAM-1 expression. To clarify the mechanisms of TNF-α-induced VCAM-1 expression in HCFs may provide therapeutic strategies for heart injury and inflammatory diseases.

Interleukin-1β (IL-1β) plays a critical mediator in the pathogenesis of eye diseases. The implication of IL-1β in inflammatory responses has been shown to be mediated through up-regulation of inflammatory genes, including matrix metalloproteinase-9 (MMP-9). However, the detailed mechanisms of IL-1β-induced MMP-9 expression in Statens Seruminstitut Rabbit Corneal Cells (SIRCs) are largely unclear. Here, we demonstrated that in SIRCs, IL-1β induced MMP-9 promoter activity and mRNA expression associated with an increase in the secretion of pro-MMP-9. IL-1β-induced pro-MMP-9 expression and MMP-9 mRNA levels were attenuated by pretreatment with the inhibitor of MEK1/2 (U0126), JNK1/2 (SP600125), NF-κB (Bay11-7082), or AP-1 (Tanshinone IIA) and transfection with siRNA of p42 or JNK2. Moreover, IL-1β markedly stimulated p42/p44 MAPK and JNK1/2 phosphorylation in SIRCs. In addition, IL-1β also enhanced p42/p44 MAPK translocation from the cytosol into the nucleus. On the other hand, IL-1β induced c-Jun and c-Fos mRNA expression, c-Jun phosphorylation, and AP-1 promoter activity. NF-κB translocation, IκBα degradation, and NF-κB promoter activity were also enhanced by IL-1β. Pretreatment with U0126 or SP600125 inhibited IL-1β-induced AP-1 and NF-κB promoter activity, but not NF-κB translocation from the cytosol into the nucleus. Finally, we established that IL-1β could stimulate SIRCs migration via p42/p44 MAPK-, JNK1/2-, AP-1-, and NF-κB-dependent MMP-9 induction. These results suggested that NF-κB and AP-1 activated by JNK1/2 and p42/p44 MAPK cascade are involved in IL-1β-induced MMP-9 expression in SIRCs.