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Fabry disease is a lysosomal storage disorder characterized by the lysosomal accumulations of glycosphingolipids in a variety of cytotypes, which include endothelial cells. The disease is inherited and originates from an error in glycosphingolipid catabolism caused by insufficient a-galactosidase A activity, which causes uncontrolled progressive storage of intracellular globotriaosylceramide (Gb3) in the vasculature and extracellular accumulation of lyso-Gb3 (a deacetylated soluble form of Gb3). Necrosis can lead to inflammation, which exacerbates necrosis and creates a positive feedback loop that triggers necroinflammation. However, the role played by necroptosis, a form of programmed necrotic cell death, in the cell-to-cell inflammatory reaction between epithelial and endothelial cells is unclear. Thus, the present study was undertaken to determine whether lyso-Gb3 induces necroptosis and whether necroptosis inhibition protects endothelial dysfunction against lyso-Gb3 inflamed retinal pigment epithelial cells. We found lyso-Gb3 induced necroptosis of a retinal pigment epithelial cell line (ARPE-19) in an autophagy-dependent manner and that conditioned media (CM) from ARPE-19 cells treated with lyso-Gb3 induced the necroptosis, inflammation, and senescence of human umbilical vein endothelial cells. In addition, a pharmacological study showed CM from lyso-Gb3 treated ARPE-19 cells induced endothelial necroptosis, inflammation, and senescence were significantly inhibited by an autophagy inhibitor (3-MA) and by two necroptosis inhibitors (necrostatin and GSK-872), respectively. These results demonstrate lyso-Gb3 induces necroptosis via autophagy and suggest that lyso-Gb3 inflamed retinal pigment epithelial cells trigger endothelial dysfunction via the autophagy-dependent necroptosis pathway. This study suggests the involvement of a novel autophagy-dependent necroptosis pathway in the regulation of endothelial dysfunction in Fabry disease. [ABSTRACT FROM AUTHOR]

Background/Aims: Idiopathic pulmonary fibrosis (IPF) is a specific form of progressive and chronic interstitial lung disease of unknown cause. IPF is characterized by excessive deposition of extracellular matrix (ECM) and destructive pathological remodeling due to epithelial-tomesenchymal transition (EMT). Eventually, lung interstitium thickens and stiffens and breathing becomes difficult. It has been well established that the transforming growth factor-β1 (TGF-β1)/Smad signaling pathway plays a critical role in the pathogenesis of pulmonary fibrosis. TGF-β1-mediated activation of mitogen activated protein kinase (MAPK) family affects Smad signaling. p90RSK is a serine/threonine kinase and is activated by the extracellular signal-regulated kinase (ERK) signaling pathway. However, the roles played by p90RSK in TGF-β1 signaling and the pathogenesis of pulmonary fibrosis remain unknown. Methods: We investigated whether p90RSK regulates the pathogenesis of pulmonary fibrosis using in vitro and in vivo systems and Western blotting, real-time quantitative PCR, transcriptional activity assays and immunofluorescence studies. Results: Pharmacological inhibition of p90RSK by FMK or inhibition of p90RSK with adenoviral vector encoding a dominant negative form of p90RSK suppressed TGF-β1-induced ECM accumulation and EMT in lung epithelial cells and fibroblasts. Interestingly, FMK significantly inhibited TGF-β1-induced smad binding elementdependent transcriptional activity, but not Smad3 phosphorylation and nuclear translocation. Furthermore, in a mouse model of bleomycin-induced lung fibrosis, FMK ameliorated pulmonary fibrosis. Conclusion: These findings indicate that p90RSK plays critical roles in pulmonary fibrosis, which suggests it be viewed as a novel therapeutic target for the treatment of lung fibrosis. [ABSTRACT FROM AUTHOR]

Cilostazol is a selective inhibitor of phosphodiesterase 3 that increases intracellular cAMP levels and activates protein kinase A, thereby inhibiting vascular smooth muscle cell (VSMC) proliferation. We investigated whether AMP-activated protein kinase (AMPK) activation induced by heme oxygenase-1 (HO-1) is a mediator of the beneficial effects of cilostazol and whether cilostazol may prevent cell proliferation and reactive oxygen species (ROS) production by activating AMPK in VSMC. In the present study, we investigated VSMC with various concentrations of cilostazol. Treatment with cilostazol increased HO-1 expression and phosphorylation of AMPK in a dose- and time-dependent manner. Cilostazol also significantly decreased platelet-derived growth factor (PDGF)-induced VSMC proliferation and ROS production by activating AMPK induced by HO-1. Pharmacological and genetic inhibition of HO-1 and AMPK blocked the cilostazol-induced inhibition of cell proliferation and ROS production. These data suggest that cilostazol-induced HO-1 expression and AMPK activation might attenuate PDGF-induced VSMC proliferation and ROS production. [ABSTRACT FROM AUTHOR]

The TGF-β (transforming growth factor-β) pathway represents an important signalling pathway involved in regulating diverse biological processes, including cell proliferation, differentiation and inflammation. Despite the critical role for TGF-β in inflammatory responses, its role in regulating NF-κB (nuclear factor-κB)-dependent inflammatory responses still remains unknown. In the present study we show that TGF-β1 synergizes with proinflammatory cytokine TNF-α (tumour necrosis factor-α) to induce NF-κB activation and the resultant inflammatory response in vitro and in vivo. TGF-β1 synergistically enhances TNF-α-induced NF-κB DNA binding activity via induction of RelA acetylation. Moreover, synergistic enhancement of TNF-α-induced RelA acetylation and DNA-binding activity by TGF-β1 is mediated by PKA (protein kinase A). Thus the present study reveals a novel role for TGF-β in inflammatory responses and provides new insight into the regulation of NF-κB by TGF-β signalling. [ABSTRACT FROM AUTHOR]

Bacteraemic pneumonia is a common cause of sepsis in critically ill patients today and is characterized by dysregulation of inflammation. The genetic factors predisposing to bacteraemic pneumonia are not yet fully understood. Innate immunity is pivotal for host defence against invading bacteria, and nuclear factor-kappa B (NF-κB) is central to bacteria-induced inflammation and immune responses. The deubiquitinating enzyme CYLD has been identified as a key negative regulator for NF-κB . In the present study, we investigated the role of CYLD in innate immune response in Escherichia coli pneumonia. Upon E. coli inoculation, Cyld−/− mice were hypersusceptible to E. coli pneumonia with higher mortality. Innate immune response to E. coli was enhanced in Cyld−/− cells and mice. Cyld−/− cells exhibited enhanced NF-κB activation upon E. coli inoculation, and the enhanced NF-κB activation by E. coli was abolished by perturbing IκB kinase (IKK) signalling. Furthermore, IKK inhibitor rescued Cyld−/− mice from lethal infection during E. coli pneumonia along with reduced inflammation. Taken together, these data showed that CYLD acts as a crucial negative regulator for E. coli pneumonia by negatively regulating NF-κB. These findings provide novel insight into the regulation of bacteraemic pneumonia and related diseases and may help develop novel therapeutic strategies for these diseases. [ABSTRACT FROM AUTHOR]

Background: Toll-like receptor 2 (TLR2) plays a critical role in mediating inflammatory/immune responses against bacterial pathogens in lung. Streptococcus pneumoniae (S. pneumoniae) and nontypeable Haemophilus influenzae (NTHi) were previously reported to synergize with each other to induce inflammatory responses. Despite the relatively known intracellular signaling pathways involved in the synergistic induction of inflammation, it is still unclear if both bacterial pathogens also synergistically induce expression of surface TLR2. Results: Here we provide direct evidence that S. pneumoniae synergizes with NTHi to upregulate TLR2 expression in lung and middle ear of the mice. Pneumolysin (PLY) appears to be the major virulence factor involved in this synergism. Moreover, S. pneumoniae PLY induces TLR2 expression via a TLR4-MyD88-NF-κB-dependent signaling pathway. Interestingly, tumor suppressor CYLD acts as a negative regulator of S. pneumoniae-induced TLR2 up-regulation via negative-crosstalk with NF-κB signaling. Conclusion: Our study thus provides novel insights into the regulation of TLR2 expression in mixed bacterial infections. [ABSTRACT FROM AUTHOR]

Vascular cell adhesion molecule (VCAM) 1 plays a central role in the recruitment of inflammatory cells, and its expression is rapidly induced by proinflammatory cytokines such as TNF-α. In the present study, we show that pretreatment with rottlerin, a specific inhibitor of protein kinase C (PKC)-δ, or transient transfection with antisense PKCδ oligonucleotides significantly inhibits TNF-α-induced expression of VCAM-1, but not of intercellular adhesion molecule (ICAM)-1 in human lung epithelium A549 cells. In addition, TNF-α was shown to induce the expression of VCAM-1 in a p38 kinase-dependent manner; also, TNF-α-induced p38 kinase activation was blocked by inhibition of PKCδ, suggesting that p38 kinase is apparently situated downstream of PKCδ in the TNF-α-signaling pathway to VCAM-1 expression. Notably, inhibition of the PKCδ-p38 kinase cascade also attenuated the TNF-α-induced adhesion of neutrophils to lung epithelium and the trafficking of leukocytes across the epithelium into the airway lumen in vivo. Together, these findings indicate that signaling via PKCδ-p38 kinase-linked cascade specifically induces expression of VCAM-1 in lung epithelium in response to TNF-α and that this effect is both functionally and clinically significant. [ABSTRACT FROM AUTHOR]

Endothelial dysfunction is one of the major initiators of atherosclerosis, and diabetes is significantly involved in this process. Shear stress-induced ERK5 activation has a critical role in regulating Kruppel-like factor 2 (KLF2) expression as well as inhibiting adhesion molecule expression, which represents one of the anti-inflammatory mechanisms. Here, we report that H2O2 and AGE negatively regulate ERK5 transcriptional activity and flow-induced anti-inflammatory actions through ERK5-SUMOylation in endothelial cells. Point-mutation analysis shows that ERK5 is covalently modified by SUMOylation at two conserved sites, Lys 6 and Lys 22. ERK5 transcriptional activity is inhibited by transfection of Ubc9 (E2 conjugase) or PIAS1 (E3 ligase). Compared with wild-type ERK5, the SUMOylation defective (K6R/K22R) mutant shows more potent transcriptional activity of both ERK5 and MEF2. H2O2 and AGE induce endogenous ERK5- SUMOylation in a time- and dose-dependent manner, and K6R/K22R mutant, DN-Ubc9, and siRNA-PIAS1 reverse ERK5/MEF2 activity as well as promoter activity of KLF2. Finally PIAS1 knock down reverses the inhibitory effect of H2O2 in shear stress-induced KLF2 and eNOS protein expression. These data clearly define SUMOylation-dependent ERK5 transcriptional repression independent of kinase activity and could be one of the molecular mechanisms of diabetes-mediated endothelial dysfunction. [ABSTRACT FROM AUTHOR]