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.

IL-1ß Induces MMP-9-Dependent Brain Astrocytic Migration via Transactivation of PDGF Receptor/NADPH Oxidase 2-Derived Reactive Oxygen Species Signals.

Matrix metalloproteinase-9 (MMP-9) plays a crucial role in pathological processes of brain inflammation, injury, and neurodegeneration. Moreover, cytokines such as interleukin-1ß (IL-1ß) induce expression of several inflammatory mediators in brain astrocytes, which may be important for brain inflammatory disorders. Recent studies have implicated that increased oxidative stress may contribute to the brain injury and inflammation. However, whether IL-1ß-induced MMP-9 expression mediated through oxidative stress remains unclear. Therefore, we investigated the role of redox signals in IL-1ß-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells). Herein, we first demonstrated that reactive oxygen species (ROS) play a crucial role in ILß-induced MMP-9 expression by zymography, real-time PCR, and ROS staining in cultured RBA-1 cells. Next, IL-1ß-induced MMP-9 expression is mediated through a c-Src-mediated transactivation of PDGFR/PI3K/Akt cascade linking to p47(phox)/NADPH oxidase 2 (Nox2)/ROS signaling pathway. Nox2-dependent ROS generation led to activation of MAPKs and the downstream transcription factors NF-κB and AP-1 (i.e., ATF2), which enhanced MMP-9 promoter activity, and thereby turned on transcription of MMP-9 gene. Functionally, IL-1ß-induced MMP-9 expression promoted astrocytic migration. These results demonstrated that in RBA-1 cells, activation of NF-κB and AP-1 (ATF2) by the c-Src/PDGFR/PI3K/Akt-mediated Nox2/ROS/MAPKs signals is required for upregulation of MMP-9 and cell migration enhanced by IL-1ß.

BK Induces cPLA2 Expression via an Autocrine Loop Involving COX-2-Derived PGE2 in Rat Brain Astrocytes.

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.

TNF-α mediates PKCδ/JNK1/2/c-Jun-dependent monocyte adhesion via ICAM-1 induction in human retinal pigment epithelial cells.

Retinal inflammatory diseases induced by cytokines, such as tumor necrosis factor-α (TNF-α) are associated with an up-regulation of intercellular adhesion molecule-1 (ICAM-1) in the retinal pigment epithelial cells (RPECs). Retinal pigment epithelium (RPE) is a monolayer of epithelial cells that forms the outer blood-retinal barrier in the posterior segment of the eye, and is also implicated in the pathology of, such as neovascularization in age-related macular degeneration (AMD). However, the detailed mechanisms of TNF-α-induced ICAM-1 expression are largely unclear in human RPECs. We demonstrated that in RPECs, TNF-α could induce ICAM-1 protein and mRNA expression and promoter activity, and monocyte adhesion. TNF-α-mediated responses were attenuated by pretreatment with the inhibitor of PKCs (Ro318220), PKCδ (Rottlerin), MEK1/2 (U0126), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA) and transfection with siRNA of TNFR1, TRAF2, JNK2, p42, or c-Jun. We showed that TNF-α could stimulate the TNFR1 and TRAF2 complex formation. TNF-α-stimulated JNK1/2 was also reduced by Rottlerin or SP600125. However, Rottlerin had no effect on TNF-α-induced p42/p44 MAPK phosphorylation. We observed that TNF-α induced c-Jun phosphorylation which was inhibited by Rottlerin or SP600125. On the other hand, TNF-α-stimulated ICAM-1 promoter activity was prominently lost in RPECs transfected with the point-mutated AP-1 ICAM-1 promoter plasmid. These results suggest that TNF-α-induced ICAM-1 expression and monocyte adhesion is mediated through a TNFR1/TRAF2/PKCδ/JNK1/2/c-Jun pathway in RPECs. These findings concerning TNF-α-induced ICAM-1 expression in RPECs imply that TNF-α might play an important role in ocular inflammation and diseases.