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.

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.

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.

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.

Lysophosphatidylcholine-induced mitochondrial fission contributes to collagen production in human cardiac fibroblasts.

Lysophosphatidylcholine (LPC) may accumulate in the heart to cause fibrotic events, which is mediated through fibroblast activation and collagen accumulation. Here, we evaluated the mechanisms underlying LPC-mediated collagen induction via mitochondrial events in human cardiac fibroblasts (HCFs), coupling application of the pharmacologic cyclooxygenase-2 (COX-2) inhibitor, celecoxib, and genetic mutations in FOXO1 on the fibrosis pathway. In HCFs, LPC caused prostaglandin E2 (PGE2)/PGE2 receptor 4 (EP4)-dependent collagen induction via activation of transcriptional activity of forkhead box protein O1 (FoxO1) on COX-2 gene expression. These responses were mediated through LPC-induced generation of mitochondrial reactive oxygen species (mitoROS), as confirmed by ex vivo studies, which indicated that LPC increased COX-2 expression and oxidative stress. LPC-induced mitoROS mediated the activation of protein kinase C (PKC)α, which interacted with and phosphorylated dynamin-related protein 1 (Drp1) at Ser616, thereby increasing Drp1-mediated mitochondrial fission and mitochondrial depolarization. Furthermore, inhibition of PKCα and Drp1 reduced FoxO1-mediated phosphorylation at Ser256 and nuclear accumulation, which suppressed COX-2/PGE2 expression and collagen production. Moreover, pretreatment with celecoxib or COX-2 siRNA suppressed WT FoxO1; mutated Ser256-to-Asp256 FoxO1-enhanced collagen induction, which was reversed by addition of PGE2 Our results demonstrate that LPC-induced generation of mitoROS regulates PKCα-mediated Drp1-dependent mitochondrial fission and COX-2 expression via a PKCα/Drp1/FoxO1 cascade, leading to PGE2/EP4-mediated collagen induction. These findings provide new insights about the role of LPC in the pathway of fibrotic injury in HCFs.

Lysophosphatidylcholine induces cyclooxygenase-2-dependent IL-6 expression in human cardiac fibroblasts.

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.

Carbon Monoxide Releasing Molecule-2-Upregulated ROS-Dependent Heme Oxygenase-1 Axis Suppresses Lipopolysaccharide-Induced Airway Inflammation.

The up-regulation of heme oxygenase-1 (HO-1) is mediated through nicotinamaide adenine dinucleotide phosphate (NADPH) oxidases (Nox) and reactive oxygen species (ROS) generation, which could provide cytoprotection against inflammation. However, the molecular mechanisms of carbon monoxide-releasing molecule (CORM)-2-induced HO-1 expression in human tracheal smooth muscle cells (HTSMCs) remain unknown. Here, we found that pretreatment with CORM-2 attenuated the lipopolysaccharide (LPS)-induced intercellular adhesion molecule (ICAM-1) expression and leukocyte count through the up-regulation of HO-1 in mice, which was revealed by immunohistochemistrical staining, Western blot, real-time PCR, and cell count. The inhibitory effects of HO-1 by CORM-2 were reversed by transfection with HO-1 siRNA. Next, Western blot, real-time PCR, and promoter activity assay were performed to examine the HO-1 induction in HTSMCs. We found that CORM-2 induced HO-1 expression via the activation of protein kinase C (PKC)α and proline-rich tyrosine kinase (Pyk2), which was mediated through Nox-derived ROS generation using pharmacological inhibitors or small interfering ribonucleic acids (siRNAs). CORM-2-induced HO-1 expression was mediated through Nox-(1, 2, 4) or p47phox, which was confirmed by transfection with their own siRNAs. The Nox-derived ROS signals promoted the activities of extracellular signal-regulated kinase 1/2 (ERK1/2). Subsequently, c-Fos and c-Jun-activator protein-1 (AP-1) subunits-were up-regulated by activated ERK1/2, which turned on transcription of the HO-1 gene by regulating the HO-1 promoter. These results suggested that in HTSMCs, CORM-2 activates PKCα/Pyk2-dependent Nox/ROS/ERK1/2/AP-1, leading to HO-1 up-regulation, which suppresses the lipopolysaccharide (LPS)-induced airway inflammation.

RTA 408 Inhibits Interleukin-1ß-Induced MMP-9 Expression via Suppressing Protein Kinase-Dependent NF-κB and AP-1 Activation in Rat Brain Astrocytes.

Neuroinflammation is characterized by the elevated expression of various inflammatory proteins, including matrix metalloproteinases (MMPs), induced by various pro-inflammatory mediators, which play a critical role in neurodegenerative disorders. Interleukin-1ß (IL-1ß) has been shown to induce the upregulation of MMP-9 through nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)-reactive oxygen species (ROS)-dependent signaling pathways. N-(2-cyano-3,12-dioxo-28-noroleana-1,9(11)-dien-17-yl)-2-2-difluoropropanamide (RTA 408), a novel synthetic triterpenoid, has been shown to possess anti-oxidant and anti-inflammatory properties in various types of cells. Here, we evaluated the effects of RTA 408 on IL-1ß-induced inflammatory responses by suppressing MMP-9 expression in a rat brain astrocyte (RBA-1) line. IL-1ß-induced MMP-9 protein and mRNA expression, and promoter activity were attenuated by RTA 408. The increased level of ROS generation in RBA-1 cells exposed to IL-1ß was attenuated by RTA 408, as determined by using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and CellROX. In addition, the inhibitory effects of RTA 408 on MMP-9 expression resulted from the suppression of the IL-1ß-stimulated activation of Pyk2 (proline-rich tyrosine kinase), platelet-derived growth factor receptor ß (PDGFRß), Akt, ROS, and mitogen-activated protein kinases (MAPKs). Pretreatment with RTA 408 attenuated the IL-1ß-induced c-Jun phosphorylation, mRNA expression, and promoter activity. IL-1ß-stimulated nuclear factor-κB (NF-κB) p65 phosphorylation, translocation, and promoter activity were also attenuated by RTA 408. Furthermore, IL-1ß-induced glial fibrillary acidic protein (GFAP) protein and mRNA expression, and cell migration were attenuated by pretreatment with RTA 408. These results provide new insights into the mechanisms by which RTA 408 attenuates IL-1ß-mediated inflammatory responses and exerts beneficial effects for the management of brain diseases.

Lipopolysaccharide-Induced Matrix Metalloproteinase-9 Expression Associated with Cell Migration in Rat Brain Astrocytes.

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.

Galangin Inhibits Thrombin-Induced MMP-9 Expression in SK-N-SH Cells via Protein Kinase-Dependent NF-κB Phosphorylation.

Galangin, a member of the flavonol compounds of the flavonoids, could exert anti-inflammatory effects in various cell types. It has been used for the treatment of arthritis, airway inflammation, stroke, and cognitive impairment. Thrombin, one of the regulators of matrix metalloproteinase (MMPs), has been known as a vital factor of physiological and pathological processes, including cell migration, the blood⁻brain barrier breakdown, brain edema formation, neuroinflammation, and neuronal death. MMP-9 especially may contribute to neurodegenerative diseases. However, the effect of galangin in combating thrombin-induced MMP-9 expression is not well understood in neurons. Therefore, we attempted to explore the molecular mechanisms by which galangin inhibited MMP-9 expression and cell migration induced by thrombin in SK-N-SH cells (a human neuroblastoma cell line). Gelatin zymography, western blot, real-time PCR, and cell migration assay were used to elucidate the inhibitory effects of galangin on the thrmbin-mediated responses. The results showed that galangin markedly attenuated the thrombin-stimulated phosphorylation of proto-oncogene tyrosine-protein kinase (c-Src), proline-rich tyrosine kinase 2 (Pyk2), protein kinase C (PKC)α/ß/δ, protein kinase B (Akt), mammalian target of rapamycin (mTOR), p42/p44 mitogen-activated protein kinase (MAPK), Jun amino-terminal kinases (JNK)1/2, p38 MAPK, forkhead box protein O1 (FoxO1), p65, and c-Jun and suppressed MMP-9 expression and cell migration in SK-N-SH cells. Our results concluded that galangin blocked the thrombin-induced MMP-9 expression in SK-N-SH cells via inhibiting c-Src, Pyk2, PKCα/ßII/δ, Akt, mTOR, p42/p44 MAPK, JNK1/2, p38 MAPK, FoxO1, c-Jun, and p65 phosphorylation and ultimately attenuated cell migration. Therefore, galangin may be a potential candidate for the management of brain inflammatory diseases.

Resveratrol Attenuates Staphylococcus Aureus-Induced Monocyte Adhesion through Downregulating PDGFR/AP-1 Activation in Human Lung Epithelial Cells.

Staphylococcus aureus (S. aureus) is a very common Gram-positive bacterium. It is widely distributed in air, soil, and water. S. aureus often causes septicemia and pneumonia in patients. In addition, it is considered to play a key role in mediating cell adhesion molecules upregulation. Resveratrol is a natural antioxidant with diverse biological effects, including the modulation of immune function, anti-inflammation, and cancer chemoprevention. In this study, we proved that S. aureus-upregulated vascular cell adhesion molecule-1 (VCAM-1) expression in human lung epithelial cells (HPAEpiCs) was inhibited by resveratrol. We also observed that resveratrol downregulated S. aureus-enhanced leukocyte count in bronchoalveolar lavage (BAL) fluid in mice. In HPAEpiCs, S. aureus stimulated c-Src, PDGFR, p38 MAPK, or JNK1/2 phosphorylation, which was inhibited by resveratrol. S. aureus induced the adhesion of THP-1 cells (a human monocytic cell line) to HPAEpiCs, which was also reduced by resveratrol. Finally, we found that S. aureus induced c-Src/PDGFR/p38 MAPK and JNK1/2-dependent c-Jun and ATF2 activation and in vivo binding of c-Jun and ATF2 to the VCAM-1 promoter, which were inhibited by resveratrol. Thus, resveratrol functions as a suppressor of S. aureus-induced inflammatory signaling, not only by inhibiting VCAM-1 expression but also by diminishing c-Src, PDGFR, JNK1/2, p38 MAPK, and AP-1 activation in HPAEpiCs.

Lipopolysaccharide induces ICAM-1 expression via a c-Src/NADPH oxidase/ROS-dependent NF-κB pathway in human pulmonary alveolar epithelial cells.

Upregulation of intercellular adhesion molecule-1 (ICAM-1) is frequently implicated in lung inflammation. Lipopolysaccharide (LPS) has been shown to play a key role in inflammation via adhesion molecule induction and then causes lung injury. However, the mechanisms underlying LPS-induced ICAM-1 expression in human pulmonary alveolar epithelial cells (HPAEpiCs) remain unclear. We showed that LPS induced ICAM-1 expression in HPAEpiCs, revealed by Western blotting, RT-PCR, real-time PCR, and promoter assay. Pretreatment with the inhibitor of c-Src (protein phosphatase-1, PP1), reactive oxygen species (ROS) (Edaravone), NADPH oxidase (apocynin and diphenyleneiodonium chloride), EGFR (AG1478), PDGFR (AG1296), phosphatidylinositol-3-kinase (PI3K) (LY294002), MEK1/2 (U0126), or NF-κB (Bay11-7082) and transfection with siRNAs of c-Src, EGFR, PDGFR, Akt, p47(phox), Nox2, Nox4, p42, and p65 markedly reduced LPS-induced ICAM-1 expression and monocyte adherence to HPAEpiCs challenged with LPS. In addition, we established that LPS stimulated phosphorylation of c-Src, EGFR, PDGFR, Akt, or p65, which was inhibited by pretreatment with their respective inhibitors. LPS induced Toll-like receptor 4 (TLR4), MyD88, TNF receptor-associated factor 6 (TRAF6), c-Src, p47(phox), and Rac1 complex formation 2, which was attenuated by transfection with c-Src or TRAF6 siRNA. Furthermore, LPS markedly enhanced NADPH oxidase activation and intracellular ROS generation, which were inhibited by PP1. We established that LPS induced p42/p44 MAPK activation via a c-Src/NADPH oxidase/ROS/EGFR, PDGFR/PI3K/Akt-dependent pathway in these cells. Finally, we observed that LPS significantly enhanced NF-κB and IκBα phosphorylation, NF-κB translocation, and NF-κB promoter activity, which were inhibited by PP1, Edaravone, apocynin, diphenyleneiodonium chloride, AG1478, AG1296, LY294002, or U0126. These results demonstrated that LPS induces p42/p44 MAPK activation mediated through the TLR4/MyD88/TRAF6/c-Src/NADPH oxidase/ROS/EGFR, PDGFR/PI3K/Akt pathway, which in turn initiates the activation of NF-κB and ultimately induces ICAM-1 expression in HPAEpiCs.

Sphingosine-1-phosphate mediates COX-2 expression and PGE2 /IL-6 secretion via c-Src-dependent AP-1 activation.

Sphingosine-1-phosphate (S1P) has been shown to regulate cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2 ) expression and IL-6 secretion in various respiratory diseases. However, the mechanisms underlying S1P-induced COX-2 expression and PGE2 production in human tracheal smooth muscle cells (HTSMCs) remain unclear. Here we demonstrated that S1P markedly induced COX-2 expression. S1P also induced PGE2 and IL-6 secretion which were reduced by the inhibitors of COX-2 (NS-398 and celecoxib). Pretreatment with the inhibitor of S1PR1 (W123), S1PR3 (CAY10444), c-Src (PP1), PYK2 (PF431396), MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA) and transfection with siRNA of S1PR1, S1PR3, c-Src, PYK2, p38, p42, JNK2, c-Jun, or c-Fos reduced S1P-induced COX-2 expression and PGE2 /IL-6 secretion. Moreover, S1P induced c-Src, PYK2, p42/p44 MAPK, JNK1/2, p38 MAPK, and c-Jun phosphorylation. We observed that S1P-induced p42/p44 MAPK and JNK1/2, but not p38 MAPK activation was mediated via a c-Src/PYK2-dependent pathway. S1P also enhanced c-Fos, but not c-Jun mRNA and protein expression and the AP-1 promoter activity. S1P-induced c-Fos mRNA and protein expression, c-Jun phosphorylation, and AP-1 promoter activity was reduced by W123, CAY10444, PP1, PF431396, U0126, SP600125, or SB202190. These results demonstrated that S1P-induced COX-2 expression and PGE2 /IL-6 generation was mediated through S1PR1/3/c-Src/PYK2/p42/p44 MAPK- or JNK1/2- and S1PR1/3/c-Src/p38 MAPK-dependent AP-1 activation.

c-Src-dependent transactivation of EGFR mediates CORM-2-induced HO-1 expression in human tracheal smooth muscle cells.

Carbon monoxide (CO), a reaction product of the cytoprotective heme oxygenase (HO)-1, displays an anti-inflammatory effect in various cellular injuries, but the precise mechanisms of HO-1 expression remain unknown. We used the transition metal carbonyl compound carbon monoxide-releasing molecule-2 (CORM-2) that acts as carbon monoxide donor. The effects of CORM-2 on expression of HO-1 in human tracheal smooth muscle cells (HTSMCs) were determined by Western blot, real-time PCR, and promoter activity assay. In HTSMCs, CORM-2 activated Nrf2 through the activation of a c-Src/EGFR/PI3K/Akt-dependent pathway, resulting in HO-1 expression. We showed that CORM-2-induced HO-1 protein and mRNA levels were inhibited by the inhibitor of c-Src (PP1 or SU6656), EGFR (AG1478), PI3K (LY294002), Akt (SH-5), JNK1/2 (SP600125), or p38 MAPK (SB202190) and transfection with siRNA of c-Src, EGFR, Akt, p38, JNK2, or Nrf2 in HTSMCs. We also showed that CORM-2 stimulated c-Src, EGFR, Akt, p38 MAPK, and JNK1/2 phosphorylation. CORM-2 also enhanced Nrf2 translocation from the cytosol to the nucleus and antioxidant response element (ARE) promoter activity. Moreover, CORM-2 mediated p38 MAPK and JNK1/2 activation via a c-Src/EGFR/PI3K/Akt pathway, which further enhanced Nrf2 activation and translocation. Finally, we observed that CORM-2 induced in vivo binding of Nrf2 to the HO-1 promoter. CORM-2 activates the c-Src/EGFR/PI3K/Akt/JNK1/2 and p38 MAPK pathways, which in turn trigger Nrf2 activation and ultimately induces HO-1 expression in HTSMCs. Thus, the HO-1/CO system might be potential therapeutics in airway diseases.

Endothelin-1 induces VCAM-1 expression-mediated inflammation via receptor tyrosine kinases and Elk/p300 in human tracheal smooth muscle cells.

The elevated level of endothelin-1 (ET-1) has been detected in the bronchoalveolar lavage of patients with severe asthma, acute lung injury, acute respiratory distress syndrome, and sepsis. ET-1 may affect vessel tone together with lung physiology and pathology. Vascular cell adhesion molecule-1 (VCAM-1) is one kind of adhesion molecules participating in the process of polymorphonuclear leukocyte transmigration and regulating the occurrence and amplification of tissue inflammation. However, the molecular mechanisms underlying ET-1-mediated expression of VCAM-1 on human tracheal smooth muscle cells (HTSMCs) were largely unknown. Here we reported that ET-1 stimulated expression of VCAM-1 gene on HTSMCs, which was blocked by pretreatment with the inhibitors of ET receptors, Src, matrix metalloproteinases (MMPs), epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), phosphatidylinositol 3-kinase (PI3K), AKT, MEK1/2, and p300, suggesting the participation of these signaling components in ET-1-regulated HTSMC responses. Furthermore, transfection with small-interfering RNA (siRNA) of Src, AKT, p42 mitogen-activated protein kinase (MAPK), or p300 downregulated the respective proteins and significantly attenuated ET-1-induced VCAM-1 expression. ET-1 also stimulated phosphorylation of Src, EGFR, PDGFR, AKT, p42/p44 MAPK, and Elk-1 and acetylation of histone H4 on HTSMCs. Immunoprecipitation assay showed the association between Elk-1 and p300 in the nucleus. Adhesion assay revealed that the adhesion of THP-1 to HTSMCs challenged with ET-1 was increased, which was attenuated by the inhibitors of ET receptors, Src, MMPs, EGFR, PDGFR, PI3K, AKT, p42/p44 MAPK, and p300. Taken together, these data suggested that ET-1 promotes occurrence and amplification of pathology-related airway inflammation via enhancing VCAM-1 expression in an ET receptor/Src/MMP/EGFR, PDGFR/PI3K/AKT/p42/p44 MAPK/Elk-1/p300 pathway in HTSMCs.

Induction of HO-1 by carbon monoxide releasing molecule-2 attenuates thrombin-induced COX-2 expression and hypertrophy in primary human cardiomyocytes.

Carbon monoxide (CO) is one of the cytoprotective byproducts of heme oxygenase (HO)-1 and exerts anti-inflammatory action in various models. However, the detailed mechanisms underlying CO-induced HO-1 expression in primary human cardiomyocytes remain largely unidentified. We used primary left ventricle myocytes as a model and applied CO releasing molecule (CORM)-2 to investigate the relationship of CO and HO-1 expression. We herein used Western blot, real-time PCR, promoter activity and EIA to investigate the role of HO-1 expression protecting against thrombin-mediated responses. We found that thrombin-induced COX-2 expression, PGE2 release and cardiomyocyte hypertrophy markers (increase in ANF/BNP, α-actin expression and cell surface area) was attenuated by pretreatment with CORM-2 which was partially reversed by hemoglobin (Hb) or ZnPP (an inhibitor of HO-1 activity), suggesting that HO-1/CO system may be of clinical importance to ameliorate heart failure through inhibition of inflammatory responses. CORM-2-induced HO-1 protein expression, mRNA and promoter was attenuated by pretreatment with the inhibitors of Pyk2 (PF431396), PDGFR (AG1296), PI3K (LY294002), Akt (SH-5), p38 (SB202530), JNK1/2 (SP600125), FoxO1 (AS1842856) and Sp1 (mithramycin A). The involvement of these signaling components was further confirmed by transfection with respective siRNAs, consistent with those of pharmacological inhibitors. These results suggested that CORM-2-induced HO-1 expression is mediated through a Pyk2/PDGFR/PI3K/Akt/FoxO1/Sp1-dependent manner and exerts a cytoprotective effect in human cardiomyocytes.

Tumor necrosis factor-alpha induces VCAM-1-mediated inflammation via c-Src-dependent transactivation of EGF receptors in human cardiac fibroblasts.

BACKGROUND: Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine and elevated in the regions of tissue injury and inflammatory diseases. The deleterious effects of TNF-α on fibroblasts may aggravate heart inflammation mediated through the up-regulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1). However, the mechanisms underlying TNF-α-induced VCAM-1 expression in cardiac fibroblasts remain unknown. This study aimed to investigate the roles of TNF-α in VCAM-1 expression and its effects on human cardiac fibroblasts (HCFs). RESULTS: The primary culture HCFs were used in this study. The results obtained with Western blotting, real time-quantitative PCR, and promoter activity analyses showed that TNF-α-induced VCAM-1 expression was mediated through TNF receptor (TNFR) 1-dependent gene up-regulation. Activation of TNFR1 by TNF-α transactivated c-Src-dependent EGF receptor (EGFR) linking to PI3K/Akt cascade, and then led to transcriptional activity of NF-κB. Moreover, the results of promoter reporter assay demonstrated that the phosphorylated p65 NF-κB turned on VCAM-1 gene expression. Subsequently, up-regulation of VCAM-1 promoted monocytes adhesion to HCFs challenged with TNF-α determined by cell adhesion assay. CONCLUSIONS: Taken together, these results indicate that in HCFs, activation of NF-κB by c-Src-mediated transactivation of EGFR/PI3K/Akt cascade is required for TNF-α-induced VCAM-1 expression. Finally, increased VCAM-1 enhances monocytes adhering to HCFs challenged with TNF-α. Understanding the mechanisms of VCAM-1 up-regulated by TNF-α on HCFs may provide rationally therapeutic interventions for heart injury or inflammatory diseases.

Nox2/ROS-dependent human antigen R translocation contributes to TNF-α-induced SOCS-3 expression in human tracheal smooth muscle cells.

Elevated levels of TNF-α have been detected in the airway fluids, which may induce upregulation of inflammatory proteins. Suppressors of cytokine signaling (SOCS)-3 proteins can be induced by various cytokines and negatively regulated inflammatory responses. Although TNF-α has been shown to induce SOCS-3 expression, the mechanisms underlying TNF-α-induced SOCS-3 expression in human tracheal smooth muscle cells (HTSMCs) remain unclear. Here, we showed that TNF-α induced SOCS-3 expression, which was inhibited by pretreatment with the inhibitor of transcription level (actinomycin D), translation level (cycloheximide), JNK1/2 (SP600125), MEK1/2 (U0126), NADPH oxidase (Nox; apocynin and diphenyleneiodonium chloride), or reactive oxygen species (ROS; N-acetyl-l-cysteine) and transfection with siRNA of JNK1, p47(phox), p42, Nox2, or human antigen R (HuR). In addition, TNF-α-stimulated JNK1/2 and p42/p44 MAPK phosphorylation, Nox activation, and ROS generation were inhibited by pretreatment with U0126 or SP600125 and transfection with siRNA of JNK1 or p42. We further showed that TNF-α markedly induced HuR protein expression and translocation from the nucleus to the cytosol, which could stabilize SOCS-3 mRNA. Moreover, TNF-α-enhanced HuR translocation was reduced by transfection with siRNA of p42, JNK1, or p47(phox). These results suggested that TNF-α induces SOCS-3 protein expression and mRNA stabilization via a TNFR1/JNK1/2, p42/p44 MAPK/Nox2/ROS-dependent HuR signaling in HTSMCs. Lipopolysaccharide (LPS) has been shown to play a key role in inflammation via induction of adhesion molecules and then causes airway and lung injury. Moreover, we also demonstrated that overexpression of SOCS-3 protects against LPS-induced adhesion molecules expression and airway inflammation.

TNF-α induces cytosolic phospholipase A2 expression via Jak2/PDGFR-dependent Elk-1/p300 activation in human lung epithelial cells.

Cytosolic phospholipase A2 (cPLA2) plays a pivotal role in mediating agonist-induced arachidonic acid release for prostaglandin (PG) synthesis during inflammation triggered by tumor necrosis factor-α (TNF-α). However, the mechanisms underlying TNF-α-induced cPLA2 expression in human lung epithelial cells (HPAEpiCs) were not completely understood. Here, we demonstrated that TNF-α induced cPLA2 mRNA and protein expression, promoter activity, and PGE2 secretion in HPAEpiCs. These responses induced by TNF-α were inhibited by pretreatment with the inhibitor of Jak2 (AG490), platelet-derived growth factor receptor (PDGFR) (AG1296), phosphoinositide 3 kinase (PI3K) (LY294002), or MEK1/2 (PD98059) and transfection with siRNA of Jak2, PDGFR, Akt, or p42. We showed that TNF-α markedly stimulated Jak2, PDGFR, Akt, and p42/p44 MAPK phosphorylation, which were attenuated by their respective inhibitors. Moreover, TNF-α stimulated Akt activation via a Jak2/PDGFR pathway in HPAEpiCs. In addition, TNF-α-induced p42/p44 MAPK phosphorylation was reduced by AG1296 or LY294002. On the other hand, TNF-α could induce Akt and p42/p44 MAPK translocation from the cytosol into the nucleus, which was inhibited by AG490, AG1296, or LY294002. Finally, we showed that TNF-α stimulated Elk-1 phosphorylation, which was reduced by LY294002 or PD98059. We also observed that TNF-α time dependently induced p300/Elk-1 and p300/Akt complex formation in HPAEpiCs, which was reduced by AG490, AG1296, or LY294002. The activity of cPLA2 protein upregulated by TNF-α was reflected on the PGE2 release, which was reduced by AG490, AG1296, LY294002, or PD98059. Taken together, these results demonstrated that TNF-α-induced cPLA2 expression and PGE2 release were mediated through a Jak2/PDGFR/PI3K/Akt/p42/p44 MAPK/Elk-1 pathway in HPAEpiCs.

Thrombin induces ICAM-1 expression in human lung epithelial cells via c-Src/PDGFR/PI3K/Akt-dependent NF-κB/p300 activation.

Up-regulation of ICAM-1 (intercellular adhesion molecule-1) is frequently implicated in lung inflammation and lung diseases, such as IPF (idiopathic pulmonary fibrosis). Thrombin has been shown to play a key role in inflammation via the induction of adhesion molecules, which then causes lung injury. However, the mechanisms underlying thrombin-induced ICAM-1 expression in HPAEpiCs (human pulmonary alveolar epithelial cells) remain unclear. In the present study, we have shown that thrombin induced ICAM-1 expression in HPAEpiCs. Pre-treatment with the inhibitor of thrombin [PPACK (D-Phe-Pro-Arg-chloromethyl ketone)], c-Src (PP1), PDGFR (platelet-derived growth factor receptor) (AG1296), PI3K (phosohinositide 3-kinase) (LY294002), NF-κB (nuclear factor κB) (Bay11-7082) or p300 (GR343) and transfection with siRNAs of c-Src, PDGFR, Akt, p65 and p300 markedly reduced thrombin-induced ICAM-1 expression and monocyte adherence to HPAEpiCs challenged with thrombin. In addition, we established that thrombin stimulated the phosphorylation of c-Src, PDGFR, Akt and p65, which were inhibited by pre-treatment with their respective inhibitors PP1, AG1296, LY294002 or Bay11-7082. In addition, thrombin also enhanced Akt and NF-κB translocation from the cytosol to the nucleus, which was reduced by PP1, AG1296 or LY294002. Thrombin induced NF-κB promoter activity and the formation of the p65-Akt-p300 complex, which were inhibited by AG1296, LY294002 or PP1. Finally, we have shown that thrombin stimulated in vivo binding of p300, Akt and p65 to the ICAM-1 promoter, which was reduced by AG1296, LY294002, SH-5 or PP1. These results show that thrombin induced ICAM-1 expression and monocyte adherence via a c-Src/PDGFR/PI3K/Akt/NF-κB-dependent pathway in HPAEpiCs. Increased understanding of the signalling mechanisms underlying ICAM-1 gene regulation will create opportunities for the development of anti-inflammatory therapeutic strategies.