Silica Nanoparticles Shed Light on Intriguing Cellular Pathways in Human Tracheal Smooth Muscle Cells: Revealing COX-2/PGE2 Production through the EGFR/Pyk2 Signaling Axis.

The use of manufactured silica nanoparticles (SiNPs) has become widespread in everyday life, household products, and various industrial applications. While the harmful effects of crystalline silica on the lungs, known as silicosis or chronic pulmonary diseases, are well understood, the impact of SiNPs on the airway is not fully explored. This study aimed to investigate the potential effects of SiNPs on human tracheal smooth muscle cells (HTSMCs). Our findings revealed that SiNPs induced the expression of cyclooxygenase-2 (COX-2) mRNA/protein and the production of prostaglandin E2 (PGE2) without causing cytotoxicity. This induction was transcription-dependent, as confirmed by cell viability assays and COX-2 luciferase reporter assays. Further analysis, including Western blot with pharmacological inhibitors and siRNA interference, showed the involvement of receptor tyrosine kinase (RTK) EGF receptor (EGFR), non-RTK Pyk2, protein kinase Cα (PKCα), and p42/p44 MAPK in the induction process. Notably, EGFR activation initiated cellular signaling that led to NF-κB p65 phosphorylation and translocation into the cell nucleus, where it bound and stimulated COX-2 gene transcription. The resulting COX-2 protein triggered PGE2 production and secretion into the extracellular space. Our study demonstrated that SiNPs mediate COX-2 up-regulation and PGE2 secretion in HTSMCs through the sequential activation of the EGFR/Pyk2/PKCα/p42/p44MAPKs-dependent NF-κB signaling pathway. Since PGE2 can have both physiological bronchodilatory and anti-inflammatory effects, as well as pathological pro-inflammatory effects, the increased PGE2 production in the airway might act as a protective compensatory mechanism and/or a contributing factor during airway exposure to SiNPs.

Rhamnetin Prevents Bradykinin-Induced Expression of Matrix Metalloproteinase-9 in Rat Brain Astrocytes by Suppressing Protein Kinase-Dependent AP-1 Activation.

Bradykinin (BK) has been recognized as a stimulant for matrix metalloproteinase (MMP)-9 expression, contributing to neuroinflammation. Modulating the BK/MMP-9 pathway offers potential in the treatment of neuroinflammatory disorders. Rhamnetin (RNT), a flavonoid compound known for its antioxidant and anti-inflammatory effects, has shown promise. However, the specific mechanisms through which RNT inhibits BK-induced MMP-9 expression remain unclear. Therefore, this study aims to delve into the intricate mechanisms underlying this process. Here, we initially demonstrated that RNT effectively attenuated BK-induced MMP-9 expression and its associated cell migration in rat brain astrocyte-1 (RBA-1) cells. Further investigation revealed that BK-driven MMP-9 protein, mRNA, and promoter activity linked to cell migration relied on c-Src, Pyk2, EGFR, PDGFR, PI3K/Akt, JNK1/2, and c-Jun. This was validated by the inhibition of these effects through specific inhibitors, a finding substantiated by the introduction of siRNAs targeting these signaling molecules. Notably, the phosphorylated levels of these signaling components induced by BK were significantly reduced by their respective inhibitors and RNT, underscoring the inhibitory role of RNT in this process. These findings indicate that, in RBA-1 cells, RNT diminishes the heightened induction of MMP-9 triggered by BK through the inhibition of c-Src/Pyk2/PDGFR and EGFR/PI3K/Akt/JNK1/2-dependent AP-1 activation. This suggests that RNT holds promise as a potential therapeutic approach for addressing neuroinflammation in the brain.

Thrombin-Induced COX-2 Expression and PGE2 Synthesis in Human Tracheal Smooth Muscle Cells: Role of PKCδ/Pyk2-Dependent AP-1 Pathway Modulation.

In this study, we confirmed that thrombin significantly increases the production of COX-2 and PGE2 in human tracheal smooth muscle cells (HTSMCs), leading to inflammation in the airways and lungs. These molecules are well-known contributors to various inflammatory diseases. Here, we investigated in detail the involved signaling pathways using specific inhibitors and small interfering RNAs (siRNAs). Our results demonstrated that inhibitors targeting proteins such as protein kinase C (PKC)δ, proline-rich tyrosine kinase 2 (Pyk2), c-Src, epidermal growth factor receptor (EGFR), phosphatidylinositol 3-kinase (PI3K), or activator protein-1 (AP-1) effectively reduced thrombin-induced COX-2 and PGE2 production. Additionally, transfection with siRNAs against PKCδ, Pyk2, c-Src, EGFR, protein kinase B (Akt), or c-Jun mitigated these responses. Furthermore, our observations revealed that thrombin stimulated the phosphorylation of key components of the signaling cascade, including PKCδ, Pyk2, c-Src, EGFR, Akt, and c-Jun. Thrombin activated COX-2 promoter activity through AP-1 activation, a process that was disrupted by a point-mutated AP-1 site within the COX-2 promoter. Finally, resveratrol (one of the most researched natural polyphenols) was found to effectively inhibit thrombin-induced COX-2 expression and PGE2 release in HTSMCs through blocking the activation of Pyk2, c-Src, EGFR, Akt, and c-Jun. In summary, our findings demonstrate that thrombin-induced COX-2 and PGE2 generation involves a PKCδ/Pyk2/c-Src/EGFR/PI3K/Akt-dependent AP-1 activation pathway. This study also suggests the potential use of resveratrol as an intervention for managing airway inflammation.

Reactive Oxygen Species-Dependent Activation of EGFR/Akt/p38 Mitogen-Activated Protein Kinase and JNK1/2/FoxO1 and AP-1 Pathways in Human Pulmonary Alveolar Epithelial Cells Leads to Up-Regulation of COX-2/PGE2 Induced by Silica Nanoparticles.

The risk of lung exposure to silica nanoparticles (SiNPs) and related lung inflammatory injury is increasing with the wide application of SiNPs in a variety of industries. A growing body of research has revealed that cyclooxygenase (COX)-2/prostaglandin E2 (PGE2) up-regulated by SiNP toxicity has a role during pulmonary inflammation. The detailed mechanisms underlying SiNP-induced COX-2 expression and PGE2 synthesis remain unknown. The present study aims to dissect the molecular components involved in COX-2/PGE2 up-regulated by SiNPs in human pulmonary alveolar epithelial cells (HPAEpiCs) which are one of the major targets while SiNPs are inhaled. In the present study, we demonstrated that SiNPs induced COX-2 expression and PGE2 release, which were inhibited by pretreatment with a reactive oxygen species (ROS) scavenger (edaravone) or the inhibitors of proline-rich tyrosine kinase 2 (Pyk2, PF-431396), epidermal growth factor receptor (EGFR, AG1478), phosphatidylinositol 3-kinase (PI3K, LY294002), protein kinase B (Akt, Akt inhibitor VIII), p38 mitogen-activated protein kinase (MAPK) (p38 MAPK inhibitor VIII), c-Jun N-terminal kinases (JNK)1/2 (SP600125), Forkhead Box O1 (FoxO1, AS1842856), and activator protein 1 (AP-1, Tanshinone IIA). In addition, we also found that SiNPs induced ROS-dependent Pyk2, EGFR, Akt, p38 MAPK, and JNK1/2 activation in these cells. These signaling pathways induced by SiNPs could further cause c-Jun and FoxO1 activation and translocation from the cytosol to the nucleus. AP-1 and FoxO1 activation could increase COX-2 and PGE2 levels induced by SiNPs. Finally, the COX-2/PGE2 axis might promote the inflammatory responses in HPAEpiCs. In conclusion, we suggested that SiNPs induced COX-2 expression accompanied by PGE2 synthesis mediated via ROS/Pyk2/EGFR/PI3K/Akt/p38 MAPK- and JNK1/2-dependent FoxO1 and AP-1 activation in HPAEpiCs.

Tumor Necrosis Factor-α-Induced C-C Motif Chemokine Ligand 20 Expression through TNF Receptor 1-Dependent Activation of EGFR/p38 MAPK and JNK1/2/FoxO1 or the NF-κB Pathway in Human Cardiac Fibroblasts.

Tumor necrosis factor (TNF)-α is involved in the pathogenesis of cardiac injury, inflammation, and apoptosis. It is a crucial pro-inflammatory cytokine in many heart disorders, including chronic heart failure and ischemic heart disease, contributing to cardiac remodeling and dysfunction. The implication of TNF-α in inflammatory responses in the heart has been indicated to be mediated through the induction of C-C Motif Chemokine Ligand 20 (CCL20). However, the detailed mechanisms of TNF-α-induced CCL20 upregulation in human cardiac fibroblasts (HCFs) are not completely defined. We demonstrated that in HCFs, TNF-α induced CCL20 mRNA expression and promoter activity leading to an increase in the secretion of CCL20. TNF-α-mediated responses were attenuated by pretreatment with TNFR1 antibody, the inhibitor of epidermal growth factor receptor (EGFR) (AG1478), p38 mitogen-activated protein kinase (MAPK) (p38 inhibitor VIII, p38i VIII), c-Jun amino N-terminal kinase (JNK)1/2 (SP600125), nuclear factor kappaB (NF-κB) (helenalin), or forkhead box O (FoxO)1 (AS1841856) and transfection with siRNA of TNFR1, EGFR, p38α, JNK2, p65, or FoxO1. Moreover, TNF-α markedly induced EGFR, p38 MAPK, JNK1/2, FoxO1, and NF-κB p65 phosphorylation which was inhibited by their respective inhibitors in these cells. In addition, TNF-α-enhanced binding of FoxO1 or p65 to the CCL20 promoter was inhibited by p38i VIII, SP600125, and AS1841856, or helenalin, respectively. Accordingly, in HCFs, our findings are the first to clarify that TNF-α-induced CCL20 secretion is mediated through a TNFR1-dependent EGFR/p38 MAPK and JNK1/2/FoxO1 or NF-κB cascade. We demonstrated that TNFR1-derived EGFR transactivation is involved in the TNF-α-induced responses in these cells. Understanding the regulation of CCL20 expression by TNF-α on HCFs may provide a potential therapeutic strategy in cardiac inflammatory disorders.

Thrombin Induces COX-2 and PGE2 Expression via PAR1/PKCalpha/MAPK-Dependent NF-kappaB Activation in Human Tracheal Smooth Muscle Cells.

The inflammation of the airway and lung could be triggered by upregulation cyclooxygenase (COX)-2 and prostaglandin E2 (PGE2) induced by various proinflammatory factors. COX-2 induction by thrombin has been shown to play a vital role in various inflammatory diseases. However, in human tracheal smooth muscle cells (HTSMCs), how thrombin enhanced the levels of COX-2/PGE2 is not completely characterized. Thus, in this study, the levels of COX-2 expression and PGE2 synthesis induced by thrombin were determined by Western blot, promoter-reporter assay, real-time PCR, and ELISA kit. The various signaling components involved in the thrombin-mediated responses were differentiated by transfection with siRNAs and selective pharmacological inhibitors. The role of NF-κB was assessed by a chromatin immunoprecipitation (ChIP) assay, immunofluorescent staining, as well as Western blot. Our results verified that thrombin markedly triggered PGE2 secretion via COX-2 upregulation which were diminished by the inhibitor of thrombin (PPACK), PAR1 (SCH79797), Gi/o protein (GPA2), Gq protein (GPA2A), PKCα (Gö6976), p38 MAPK (SB202190), JNK1/2 (SP600125), MEK1/2 (U0126), or NF-κB (helenalin) and transfection with siRNA of PAR1, Gq α, Gi α, PKCα, JNK2, p38, p42, or p65. Moreover, thrombin induced PAR1-dependent PKCα phosphorylation in HTSMCs. We also observed that thrombin induced p38 MAPK, JNK1/2, and p42/p44 MAPK activation through a PAR1/PKCα pathway. Thrombin promoted phosphorylation of NF-κB p65, leading to nuclear translocation and binding to the COX-2 promoter element to enhance promoter activity, which was reduced by Gö6976, SP600125, SB202190, or U0126. These findings supported that COX-2/PGE2 expression triggered by thrombin was engaged in PAR1/Gq or Gi/o/PKCα/MAPK-dependent NF-κB activation in HTSMCs.

Induction of Heme Oxygenase-1 by 15d-Prostaglandin J2 Mediated via a ROS-Dependent Sp1 and AP-1 Cascade Suppresses Lipopolysaccharide-Triggered Interleukin-6 Expression in Mouse Brain Microvascular Endothelial Cells.

Heme oxygenase-1 (HO-1) has been shown to exert antioxidant, anti-inflammatory, and anti-apoptotic effects in various types of cells. Therefore, the induction of HO-1 is an excellent rationale for the development of protective drugs. 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) can modulate the expression of antioxidant defense proteins and be beneficial for neuroinflammation. Brain endothelial cells play an important role in the pathophysiology of brain disorders. Whether 15d-PGJ2 can induce HO-1 expression and protect against the inflammatory responses in mouse brain microvascular endothelial (bEnd.3) cells remains unclear. Here, we reveal that 15d-PGJ2 stimulated HO-1 protein and mRNA expression in a time- and concentration-dependent manner in bEnd.3 cells, which was attenuated by diphenyleneiodonium chloride (DPI) and MitoTempo. Thus, activation of NADPH oxidase (NOX)- and mitochondria-derived reactive oxygen species (ROS) mediated 15d-PGJ2-induced HO-1 expression. ROS generation could cause phosphorylation of protein kinase C (PKC)δ, leading to HO-1 expression, which was suppressed by Rottlerin (selective inhibitor PKCδ), DPI, and MitoTempo. We further demonstrated that phosphorylation of c-Jun N-terminal kinase (JNK)1/2 participated in 15d-PGJ2-upregulated HO-1 expression, which was blocked by SP600125 or Rottlerin. Moreover, 15d-PGJ2-induced HO-1 expression was mediated through the activation of c-Jun (a subunit of activator protein 1 (AP-1)) and specificity protein 1 (Sp1), leading to their interaction with the HO-1 promoter, revealed by chromatin immunoprecipitation assay, which was attenuated by SP600125, Mithramycin A, or Tanshinone II A. We further verified the anti-inflammatory effect of HO-1 expression. Our results showed that 15d-PGJ2-induced HO-1 could mitigate the lipopolysaccharide-triggered interleukin-6 expression and secretion, as measured by an ELISA assay kit. These results suggest that 15d-PGJ2-induced HO-1 expression is mediated through the activation of NOX- and mitochondria-derived ROS-dependent PKCδ/JNK1/2/Sp1 and the AP-1 signaling pathway and protects against inflammatory responses in bEnd.3 cells.

HO-1 Upregulation by Kaempferol via ROS-Dependent Nrf2-ARE Cascade Attenuates Lipopolysaccharide-Mediated Intercellular Cell Adhesion Molecule-1 Expression in Human Pulmonary Alveolar Epithelial Cells.

Lung inflammation is a pivotal event in the pathogenesis of acute lung injury. Heme oxygenase-1 (HO-1) is a key antioxidant enzyme that could be induced by kaempferol (KPR) and exerts anti-inflammatory effects. However, the molecular mechanisms of KPR-mediated HO-1 expression and its effects on inflammatory responses remain unknown in human pulmonary alveolar epithelial cells (HPAEpiCs). This study aimed to verify the relationship between HO-1 expression and KPR treatment in both in vitro and in vivo models. HO-1 expression was determined by real time-PCR, Western blotting, and promoter reporter analyses. The signaling components were investigated by using pharmacological inhibitors or specific siRNAs. Chromatin immunoprecipitation (ChIP) assay was performed to investigate the interaction between nuclear factor erythroid-2-related factor (Nrf2) and antioxidant response elements (ARE) binding site of HO-1 promoter. The effect of KPR on monocytes (THP-1) binding to HPAEpiCs challenged with lipopolysaccharides (LPS) was determined by adhesion assay. We found that KPR-induced HO-1 level attenuated the LPS-induced intercellular cell adhesion protein 1 (ICAM-1) expression in HPAEpiCs. KPR-induced HO-1 mRNA and protein expression also attenuated ICAM-1 expression in mice. Tin protoporphyrin (SnPP)IX reversed the inhibitory effects of KPR in HPAEpiCs. In addition, in HPAEpiCs, KPR-induced HO-1 expression was abolished by both pretreating with the inhibitor of NADPH oxidase (NOX, apocynin (APO)), reactive oxygen species (ROS) (N-acetyl-L-cysteine (NAC)), Src (Src kinase inhibitor II (Srci II)), Pyk2 (PF431396), protein kinase C (PKC)α (Gö6976), p38 mitogen-activated protein kinase (MAPK) inhibitor (p38i) VIII, or c-Jun N-terminal kinases (JNK)1/2 (SP600125) and transfection with their respective siRNAs. The transcription of the homx1 gene was enhanced by Nrf2 activated by JNK1/2 and p38α MAPK. The binding activity between Nrf2 and HO-1 promoter was attenuated by APO, NAC, Srci II, PF431396, or Gö6983. KPR-mediated NOX/ROS/c-Src/Pyk2/PKCα/p38α MAPK and JNK1/2 activate Nrf2 to bind with ARE on the HO-1 promoter and induce HO-1 expression, which further suppresses the LPS-mediated inflammation in HPAEpiCs. Thus, KPR exerts a potential strategy to protect against pulmonary inflammation via upregulation of the HO-1.

5,8-Dihydroxy-4 ', 7-dimethoxyflavone Attenuates TNF-α-Induced Expression of Vascular Cell Adhesion Molecule-1 through EGFR/PKCα/PI3K/Akt/Sp1-Dependent Induction of Heme Oxygenase-1 in Human Cardiac Fibroblasts.

Recently, we found that 5,8-dihydroxy-4',7-dimethoxyflavone (DDF) upregulated the expression of heme oxygenase (HO)-1 via p38 mitogen-activated protein kinase/nuclear factor-erythroid factor 2-related factor 2 (MAPK/Nrf2) pathway in human cardiac fibroblasts (HCFs). However, the alternative processes by which DDF induces the upregulation of HO-1 expression are unknown. Activation of epidermal growth factor receptor (EGFR), phosphoinositide 3-kinase/protein kinase B (PI3K/Akt), and protein kinase C (PKC)α may initiate specificity protein (Sp)1 activity, which has been reported to induce expression of antioxidant molecules. Thus, we explored whether these components are engaged in DDF-induced HO-1 upregulation in HCFs. Western blotting, promoter-reporter analyses, and real-time polymerase chain reactions were adopted to measure HO-1 and vascular cell adhesion molecule (VCAM)-1 expressions in HCFs. Respective small interfering (si)RNAs and pharmacological inhibitors were employed to investigate the signaling components engaged in DDF-induced HO-1 upregulation. The chromatin immunoprecipitation assay was conducted to detect the binding interaction of Sp1 and antioxidant response elements (ARE) on the promoter of HO-1. An adhesion assay of THP-1 monocyte was undertaken to examine the functional effect of HO-1 on tumor necrosis factor (TNF)-α-induced VCAM-1 expression. DDF stimulated the EGFR/PKCα/PI3K/Akt pathway leading to activation of Sp1 in HCFs. The roles of these protein kinases in HO-1 induction were ensured by transfection with their respective siRNAs. Chromatin immunoprecipitation assays revealed the interaction between Sp1 and the binding site of proximal ARE on the HO-1 promoter, which was abolished by glutathione, AG1478, Gö6976, LY294002, or mithramycin A. HO-1 expression enhanced by DDF abolished the monocyte adherence to HCFs and VCAM-1 expression induced by TNF-α. Pretreatment with an inhibitor of HO-1: zinc protoporphyrin IX reversed these inhibitory effects of HO-1. We concluded that DDF-induced HO-1 expression was mediated via an EGFR/PKCα/PI3K/Akt-dependent Sp1 pathway and attenuated the responses of inflammation in HCFs.

Sphingosine 1-Phosphate-Upregulated COX-2/PGE2 System Contributes to Human Cardiac Fibroblast Apoptosis: Involvement of MMP-9-Dependent Transactivation of EGFR Cascade.

Human cardiac fibroblasts (HCFs) play key roles in normal physiological functions and pathological processes in the heart. Our recent study has found that, in HCFs, sphingosine 1-phosphate (S1P) can upregulate the expression of cyclooxygenase-2 (COX-2) leading to prostaglandin E2 (PGE2) generation mediated by S1P receptors/PKCα/MAPKs cascade-dependent activation of NF-κB. Alternatively, G protein-coupled receptor- (GPCR-) mediated transactivation of receptor tyrosine kinases (RTKs) has been proved to induce inflammatory responses. However, whether GPCR-mediated transactivation of RTKs participated in the COX-2/PGE2 system induced by S1P is still unclear in HCFs. We hypothesize that GPCR-mediated transactivation of RTKs-dependent signaling cascade is involved in S1P-induced responses. This study is aimed at exploring the comprehensive mechanisms of S1P-promoted COX-2/PGE2 expression and apoptotic effects on HCFs. Here, we used pharmacological inhibitors and transfection with siRNA to evaluate whether matrix metalloprotease (MMP)2/9, heparin-binding- (HB-) epidermal growth factor (EGF), EGF receptor (EGFR), PI3K/Akt, MAPKs, and transcription factor AP-1 participated in the S1P-induced COX-2/PGE2 system determined by Western blotting, real-time polymerase chain reaction (RT-PCR), chromatin immunoprecipitation (ChIP), and promoter-reporter assays in HCFs. Our results showed that S1PR1/3 activated by S1P coupled to Gq- and Gi-mediated MMP9 activity to stimulate EGFR/PI3K/Akt/MAPKs/AP-1-dependent activity of transcription to upregulate COX-2 accompanied with PGE2 production, leading to stimulation of caspase-3 activity and apoptosis. Moreover, S1P-enhanced c-Jun bound to COX-2 promoters on its corresponding binding sites, which was attenuated by these inhibitors of protein kinases, determined by a ChIP assay. These results concluded that transactivation of MMP9/EGFR-mediated PI3K/Akt/MAPKs-dependent AP-1 activity was involved in the upregulation of the COX-2/PGE2 system induced by S1P, in turn leading to apoptosis in HCFs.

Anti-Inflammatory Effects of Rhamnetin on Bradykinin-Induced Matrix Metalloproteinase-9 Expression and Cell Migration in Rat Brain Astrocytes.

Bradykinin (BK) has been shown to induce matrix metalloproteinase (MMP)-9 expression and participate in neuroinflammation. The BK/MMP-9 axis can be a target for managing neuroinflammation. Our previous reports have indicated that reactive oxygen species (ROS)-mediated nuclear factor-kappaB (NF-κB) activity is involved in BK-induced MMP-9 expression in rat brain astrocytes (RBA-1). Rhamnetin (RNT), a flavonoid compound, possesses antioxidant and anti-inflammatory effects. Thus, we proposed RNT could attenuate BK-induced response in RBA-1. This study aims to approach mechanisms underlying RNT regulating BK-stimulated MMP-9 expression, especially ROS and NF-κB. We used pharmacological inhibitors and siRNAs to dissect molecular mechanisms. Western blotting and gelatin zymography were used to evaluate protein and MMP-9 expression. Real-time PCR was used for gene expression. Wound healing assay was applied for cell migration. 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) were used for ROS generation and NOX activity, respectively. Promoter luciferase assay and chromatin immunoprecipitation (ChIP) assay were applied to detect gene transcription. Our results showed that RNT inhibits BK-induced MMP-9 protein and mRNA expression, promoter activity, and cell migration in RBA-1 cells. Besides, the levels of phospho-PKCδ, NOX activity, ROS, phospho-ERK1/2, phospho-p65, and NF-κB p65 binding to MMP-9 promoter were attenuated by RNT. In summary, RNT attenuates BK-enhanced MMP-9 upregulation through inhibiting PKCδ/NOX/ROS/ERK1/2-dependent NF-κB activity in RBA-1.

Involvement of FoxO1, Sp1, and Nrf2 in Upregulation of Negative Regulator of ROS by 15d-PGJ2 Attenuates H2O2-Induced IL-6 Expression in Rat Brain Astrocytes.

Excessive production of reactive oxygen species (ROS) by NADPH oxidase (Nox) resulted in inflammation. The negative regulator of ROS (NRROS) dampens ROS generation during inflammatory responses. 15-Deoxy-∆12,14-prostaglandin J2 (15d-PGJ2) exhibits neuroprotective effects on central nervous system (CNS). However, whether 15d-PGJ2-induced NRROS expression was unknown in rat brain astrocytes (RBA-1). NRROS expression was determined by Western blot, RT/real-time PCR, and promoter activity assays. The signaling components were investigated using pharmacological inhibitors or specific siRNAs. The interaction between transcription factors and the NRROS promoter was investigated by chromatin immunoprecipitation assay. Upregulation of NRROS on the hydrogen peroxide (H2O2)-mediated ROS generation and interleukin 6 (IL-6) secretion was measured. 15d-PGJ2-induced NRROS expression was mediated through PI3K/Akt-dependent activation of Sp1 and FoxO1 and established the essential promoter regions. We demonstrated that 15d-PGJ2 activated PI3K/Akt and following by cooperation between phosphorylated nuclear FoxO1 and Sp1 to initiate the NRROS transcription. In addition, Nrf2 played a key role in NRROS expression induced by 15d-PGJ2 which was mediated through its phosphorylation. Finally, the NRROS stable clones attenuated the H2O2-induced ROS generation and expression of IL-6 through suppressing the Nox-2 activity. These results suggested that 15d-PGJ2-induced NRROS expression is mediated through a PI3K/Akt-dependent FoxO1 and Sp1 phosphorylation, and Nrf2 cascade, which suppresses ROS generation through attenuating the p47phox phosphorylation and gp91phox formation and IL-6 expression in RBA-1 cells. These results confirmed the mechanisms underlying 15d-PGJ2-induced NRROS expression which might be a potential strategy for prevention and management of brain inflammatory and neurodegenerative diseases.

Carbon Monoxide Releasing Molecule-3 Enhances Heme Oxygenase-1 Induction via ROS-Dependent FoxO1 and Nrf2 in Brain Astrocytes.

Carbon monoxide releasing molecule-3 (CORM-3) has been shown to protect inflammatory diseases via the upregulation of heme oxygenases-1 (HO-1). However, in rat brain astrocytes (RBA-1), the mechanisms underlying CORM-3-induced HO-1 remain poorly defined. This study used western blot, real-time PCR, and promoter activity assays to determine the levels of HO-1 expression and 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) and dihydroethidium (DHE) to measure reactive oxygen species (ROS). We found that CORM-3-induced HO-1 expression was mediated through ROS generation by Nox or mitochondria. The signaling components were differentiated by pharmacological inhibitors and small interfering RNA (siRNA). Subcellular fractions, immunofluorescent staining, and chromatin immunoprecipitation assay were used to evaluate the nuclear translocation and promoter binding activity of Nrf2 induced by CORM-3. The roles of mTOR and FoxO1 in CORM-3-stimulated responses are still unknown in RBA-1 cells. Our results demonstrated that transfection with siRNAs or pretreatment with pharmacological inhibitors attenuated the levels of HO-1 and phosphorylation of signaling components including Akt, mTOR, FoxO1, and Nrf2 stimulated by CORM-3. Moreover, pretreatment with N-acetyl-L-cysteine, diphenyleneiodonium chloride, apocynin, or rotenone blocked nuclear translocation and promoter binding activity of Nrf2 induced by CORM-3. The present study concluded that in RBA-1 cells, CORM-3-induced HO-1 expression is, at least partially, mediated through Nox and mitochondria/ROS-dependent PI3K/Akt/mTOR cascade to activate FoxO1 or ROS leading to activation of Nrf2 activity.

Upregulation of COX-2 and PGE2 Induced by TNF-α Mediated Through TNFR1/MitoROS/PKCα/P38 MAPK, JNK1/2/FoxO1 Cascade in Human Cardiac Fibroblasts.

PURPOSE: Tumor necrosis factor-α (TNF-α) has been shown to exert as a pathogenic factor in cardiac fibrosis and heart failure which were associated with the up-regulation of cyclooxygenase (COX)-2/prostaglandin E2 (PGE2) axis. However, whether TNF-α-induced COX-2/PGE2 upregulation mediated through ROS-dependent cascade remains elusive in human cardiac fibroblasts (HCFs). This study aims to address the underlying mechanisms of TNF-α-induced COX-2/PGE2 expression. METHODS: Here, we used TNF receptor neutralizing antibody (TNFR nAb), pharmacologic inhibitors, and siRNAs to dissect the involvement of signaling components examined by Western blot and ELISA in TNF-α-mediated responses in HCFs. MitoSOX Red was used to measure mitoROS generation. Isolation of subcellular fractions was performed to determine membrane translocation of PKCα. Promoter luciferase assay and chromatin immunoprecipitation (ChIP) assay were used to determine the role of transcription factor. RESULTS: We found that TNF-α time- and concentration-dependently upregulated COX-2 protein and mRNA expression as well as PGE2 synthesis which was attenuated by TNFR1 nAb, the inhibitor of mitochondrial ROS scavenger (MitoTEMPO), protein kinase C [(PKC)α, Gö6976], p38 MAPK [p38 inhibitor VIII, (p38i VIII)], JNK1/2 (SP600125), or forkhead box protein O1 [(FoxO1), AS1842856], and transfection with their respective siRNAs in HCFs. TNF-α-stimulated PKCα phosphorylation was inhibited by TNFR1 nAb, MitoTEMPO, or Gö6976. TNF-α stimulated phosphorylation of p38 MAPK and JNK1/2 was attenuated by TNFR1 nAb, MitoTEMPO, Gö6976, and their inhibitors p38i VIII and SP600125. Moreover, TNF-α-triggered FoxO1 phosphorylation was abolished by AS1842856, TNFR1 nAb, and its upstream inhibitors MitoTEMPO, Gö6976, p38i VIII, and SP600125. Phosphorylation of FoxO1 could enhance its interaction with the COX-2 promoter element revealed by ChIP assay, which was attenuated by AS1842856. CONCLUSION: Our results suggested that TNF-α-induced COX-2/PGE2 upregulation is mediated through TNFR1-dependent MitoROS/PKCα/p38 MAPK and JNK1/2 cascade to activate FoxO1 binding with the COX-2 promoter in HCFs.

Induction of HO-1 by 5, 8-Dihydroxy-4',7-Dimethoxyflavone via Activation of ROS/p38 MAPK/Nrf2 Attenuates Thrombin-Induced Connective Tissue Growth Factor Expression in Human Cardiac Fibroblasts.

Heme oxygenase-1 (HO-1) has been shown to exert as an antioxidant and anti-inflammatory enzyme in cardiovascular inflammatory diseases. Flavonoids have been demonstrated to display anti-inflammatory and antioxidant effects through the induction of HO-1. 5,8-Dihydroxy-4',7-dimethoxyflavone (DDF), one of the flavonoid compounds, is isolated from Reevesia formosana. Whether DDF induced HO-1 expression on human cardiac fibroblasts (HCFs) remained unknown. Here, we found that DDF time- and concentration-dependently induced HO-1 protein and mRNA expression, which was attenuated by pretreatment with reactive oxygen species (ROS) scavenger N-acetyl cysteine (NAC) in HCFs. DDF-enhanced ROS generation was attenuated by NAC, but not by either diphenyleneiodonium chloride (DPI, Nox inhibitor) or MitoTempol (mitochondrial ROS scavenger). Interestingly, pretreatment with glutathione (GSH) inhibited DDF-induced HO-1 expression. The ratio of GSH/GSSG was time-dependently decreased in DDF-treated HCFs. DDF-induced HO-1 expression was attenuated by an inhibitor of p38 MAPK (p38i VIII) or siRNA, but not by MEK1/2 (PD98059) or JNK1/2 (SP600125). DDF-stimulated p38 MAPK phosphorylation was inhibited by GSH or p38i VIII. Moreover, DDF-induced HO-1 expression was mediated through Nrf2 phosphorylation and translocation into the nucleus which was attenuated by NAC or p38 siRNA. DDF also stimulated antioxidant response element (ARE) promoter activity which was inhibited by NAC, GSH, or p38i VIII. Interaction between Nrf2 and the ARE-binding sites on the HO-1 promoter was revealed by chromatin immunoprecipitation assay, which was attenuated by NAC, GSH, or p38i VIII. We further evaluated the functional effect of HO-1 expression on the thrombin-induced fibrotic responses. Our result indicated that the induction of HO-1 by DDF can attenuate the thrombin-induced connective tissue growth factor expression. These results suggested that DDF-induced HO-1 expression is, at least, mediated through the activation of the ROS-dependent p38 MAPK/Nrf2 signaling pathway in HCFs. Thus, the upregulation of HO-1 by DDF could be a candidate for the treatment of heart fibrosis.

Sphingosine 1-Phosphate Induces Cyclooxygenase-2/Prostaglandin E2 Expression via PKCα-dependent Mitogen-Activated Protein Kinases and NF-κB Cascade in Human Cardiac Fibroblasts.

In the regions of tissue injuries and inflammatory diseases, sphingosine 1-phosphate (S1P), a proinflammatory mediator, is increased. S1P may induce the upregulation of cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) system in various types of cells to exacerbate heart inflammation. However, the detailed molecular mechanisms by which S1P induces COX-2 expression in human cardiac fibroblasts (HCFs) remain unknown. HCFs were incubated with S1P and analyzed by Western blotting, real time-Polymerase chain reaction (RT-PCR), and immunofluorescent staining. Our results indicated that S1P activated S1PR1/3-dependent transcriptional activity to induce COX-2 expression and PGE2 production. S1P recruited and activated PTX-sensitive Gi or -insensitive Gq protein-coupled S1PR and then stimulated PKCα-dependent phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK1/2, leading to activating transcription factor NF-κB. Moreover, S1P-activated NF-κB was translocated into the nucleus and bound to its corresponding binding sites on COX-2 promoters determined by chromatin immunoprecipitation (ChIP) and promoter-reporter assays, thereby turning on COX-2 gene transcription associated with PGE2 production in HCFs. These results concluded that in HCFs, activation of NF-κB by PKCα-mediated MAPK cascades was essential for S1P-induced up-regulation of the COX-2/PGE2 system. Understanding the mechanisms of COX-2 expression and PGE2 production regulated by the S1P/S1PRs system on cardiac fibroblasts may provide rationally therapeutic interventions for heart injury or inflammatory diseases.

Galangin Inhibits LPS-Induced MMP-9 Expression via Suppressing Protein Kinase-Dependent AP-1 and FoxO1 Activation in Rat Brain Astrocytes.

PURPOSE: Neuroinflammation, characterized by the increased expression of inflammatory proteins such as matrix metalloproteinases (MMPs), plays a critical role in neurodegenerative disorders. Lipopolysaccharide (LPS) has been shown to upregulate MMP-9 expression through the activation of various transcription factors, including activator protein 1 (AP-1) and forkhead box protein O1 (FoxO1). The flavonoid 3,5,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one (galangin) has been demonstrated to possess antioxidant and anti-inflammatory properties in various types of cells. Here, we investigated the mechanisms underlying the inhibitory effect of galangin on LPS-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells). METHODS: Pharmacological inhibitors and siRNAs were employed to explore the effects of galangin on LPS-challenged RBA-1 cells. Gelatin zymography, Western blotting, real-time PCR, and a luciferase reporter assay were used to detect MMP-9 activity, protein expression, mRNA levels, and promoter activity, respectively. The protein kinases involved in the LPS-induced MMP-9 expression were determined by Western blot. A chromatin immunoprecipitation (ChIP) assay was employed to evaluate the activity of c-Jun at the MMP-9 promoter. RESULTS: Galangin treatment attenuated the LPS-mediated induction of MMP-9 protein and mRNA expression, as well as the activity at the MMP-9 promoter. In addition, galangin exerted its inhibitory effects on MMP-9 expression through suppressing the LPS-stimulated activation of proline-rich tyrosine kinase (Pyk2), platelet-derived growth factor receptor beta (PDGFRß), phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), mammalian target of rapamycin (mTOR), and mitogen-activated protein kinases (MAPKs). Pretreatment with galangin attenuated the LPS-induced phosphorylation of c-Jun and FoxO1. LPS-induced cell migration was also suppressed by galangin pretreatment. CONCLUSION: Galangin attenuates the LPS-induced inflammatory responses, including the induction of MMP-9 expression and cell migration, via inhibiting Pyk2/PDGFRß/PI3K/Akt/mTOR/JNK1/JNK2 and p44/p42 MAPK cascade-dependent AP-1 and FoxO1 activities. These results provide new insights into the mechanisms through which galangin mitigates LPS-induced inflammatory responses, and suggest novel strategies for the management of LPS-related brain diseases.

Pristimerin Inhibits MMP-9 Expression and Cell Migration Through Attenuating NOX/ROS-Dependent NF-κB Activation in Rat Brain Astrocytes Challenged with LPS.

PURPOSE: Neuroinflammation plays a crucial role in neurodegenerative diseases. Matrix metalloproteinases (MMPs) are a landmark of neuroinflammation. Lipopolysaccharide (LPS) has been demonstrated to induce MMP-9 expression. The mechanisms underlying LPS-induced MMP-9 expression have not been completely elucidated in astrocytes. Nuclear factor-kappaB (NF-κB) is well known as one of the crucial transcription factors in MMP-9 induction. Moreover, reactive oxygen species (ROS) could be an important mediator of neuroinflammation. Here, we differentiated whether ROS and NF-κB contributed to LPS-mediated MMP-9 expression in rat brain astrocytes (RBA-1). Besides, pristimerin has been revealed to possess antioxidant and anti-inflammatory effects. We also evaluated the effects of pristimerin on LPS-induced inflammatory responses. METHODS: RBA-1 cells were used for analyses. Pharmacological inhibitors and siRNAs were used to evaluate the signaling pathway. Western blotting and gelatin zymography were conducted to evaluate protein and MMP-9 expression, respectively. Real-time PCR was for mRNA expression. Wound healing assay was for cell migration. 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA) and dihydroethidium (DHE) staining were for ROS generation. Immunofluorescence staining was conducted to assess NF-κB p65. Promoter-reporter gene assay and chromatin immunoprecipitation (ChIP) assay were used to detect promoter activity and the association of nuclear proteins with the promoter. RESULTS: Our results showed that the increased level of ROS generation was attenuated by edaravone (a ROS scavenger), apocynin (APO; an inhibitor of p47Phox), diphenyleneiodonium (DPI; an inhibitor of NOX), and pristimerin in RBA-1 cells exposed to LPS. Besides, pretreatment with APO, DPI, edaravone, Bay11-7082, and pristimerin also inhibited the phosphorylation, nuclear translocation, promoter binding activity of NF-κB p65 as well as upregulation of MMP-9 expression-mediated cell migration in RBA-1 cells challenged with LPS. CONCLUSION: These results suggested that LPS enhances the upregulation of MMP-9 through nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)/ROS-dependent NF-κB activity. These results also provide new insights into the mechanisms by which pristimerin attenuates LPS-mediated MMP-9 expression and neuroinflammatory responses.

Mevastatin-Induced AP-1-Dependent HO-1 Expression Suppresses Vascular Cell Adhesion Molecule-1 Expression and Monocyte Adhesion on Human Pulmonary Alveolar Epithelial Cells Challenged with TNF-α.

Mevastatin (MVS) has been previously shown to induce heme oxygenase (HO)-1 expression through Nox/ROS-dependent PDGFRα/PI3K/Akt/Nrf2/ARE axis in human pulmonary alveolar epithelial cells (HPAEpiCs). However, alternative signaling pathways might involve in MVS-induced HO-1 expression. We found that tumor necrosis factor α (TNFα) induced vascular cell adhesion protein 1 (VCAM-1) expression and NF-κB p65 phosphorylation which were attenuated by pretreatment with MVS via up-regulation of HO-1, determined by Western blot and real-time qPCR. TNFα-induced VCAM-1 expression was attenuated by an NF-κB inhibitor, Bay117082. The inhibitory effects of MVS were reversed by tin protoporphyrin (SnPP)IX (an inhibitor of HO-1 activity). In addition, pretreatment with the inhibitor of pan-Protein kinase C (PKC) (GF109203X), PKCα (Gö6983), Pyk2 (PF431396), p38α MAPK (SB202190), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA), and transfection with their respective siRNAs abolished MVS-induced HO-1 expression in HPAEpiCs. c-Jun (one of AP-1 subunits) was activated by PKCα, Pyk2, p38α MAPK, and JNK1/2, which turned on the transcription of the homx1 gene. The interaction between c-Jun and HO-1 promoter was confirmed by a chromatin immunoprecipitation (ChIP) assay, which was attenuated by these pharmacological inhibitors. These results suggested that MVS induces AP-1/HO-1 expression via PKCα/Pyk2/p38α MAPK- or JNK1/2-dependent c-Jun activation, which further binds with AP-1-binding site on HO-1 promoter and suppresses the TNFα-mediated inflammatory responses in HPAEpiCs. Thus, upregulation of the AP-1/HO-1 system by MVS exerts a potentially therapeutic strategy to protect against pulmonary inflammation.

Induction of HO-1 by Mevastatin Mediated via a Nox/ROS-Dependent c-Src/PDGFRα/PI3K/Akt/Nrf2/ARE Cascade Suppresses TNF-α-Induced Lung Inflammation.

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)-α-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)α, 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/PDGFRα/PI3K/Akt-regulated Nrf2/ARE axis and suppresses the TNF-α-mediated inflammatory responses in HPAEpiCs.