Browsing by keyword "Sphingomyelin Phosphodiesterase"
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Defective acid sphingomyelinase pathway with Pseudomonas aeruginosa infection in cystic fibrosisAcid sphingomyelinase (ASMase) is a key enzyme in sphingolipid metabolism, which can be activated by various cellular stress mechanisms including bacterial pathogens. Activation of ASMase generates ceramide, which is important for innate immune response to eliminate infected pathogens. The current study reveals a defective ASMase pathway after Pseudomonas aeruginosa infection in both a cystic fibrosis (CF) bronchial epithelial cell line (IB3-1 cell) and in the lungs of CF transmembrane conductance regulator (CFTR) knockout (KO) mice as compared with S9 cells and wild-type C57BL/6 mice. ASMase activity and total ceramide levels significantly increased in S9 cells and C57BL/6 mice with P. aeruginosa infection, but not in IB3-1 cells and CFTR KO mice. The silencing of CFTR by CFTR RNAi in S9 cells significantly decreased ASMase activity after bacterial infection as compared with controls. This study also demonstrates that induction of ASMase is responsible for modulating the immune response to bacterial infection. Blocking ASMase activity with specific ASMase RNAi, an ASMase inhibitor, or an ASMase antibody in S9 cells significantly increased IL-8 levels with P. aeruginosa infection compared with controls. Reciprocally, adding exogenous bacterial sphingomyelinase to IB3-1 cells significantly decreased IL-8 levels compared with untreated cells. In addition, silencing of ASMase in S9 cells also significantly decreased bacterial internalization. Adding exogenous bacterial sphingomyelinase to IB3-1 cells reconstituted the cell death response to P. aeruginosa infection. This study demonstrates that the defective ASMase pathway in CF is a key contributor to the unabated IL-8 response with P. aeruginosa infection and to the compromised host response failing to eradicate bacteria.
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Effects of ceramide and protein kinase C on the regulation of type I 5'-deiodinase in FRTL-5 rat thyroid cellsTumor necrosis factor-alpha (TNF alpha) is a pleiotropic cytokine that is involved in the regulation of thyroid growth and differentiated functions. The cellular and molecular mechanisms involved in mediating the effects of TNF alpha on thyroid function, however, are unknown. In a number of cell types, TNF alpha receptor binding results in the activation of specific signal transduction cascades, including protein kinase C (PKC) and the hydrolysis of sphingomyelin to ceramide. In the present study, we examined the possible role of PKC and the hydrolysis of sphingomyelin to ceramide in the regulation of TSH-induced increases in 5'-deiodinase (5'D-I) activity and 5'D-I messenger RNA (mRNA) levels in FRTL-5 cells. Further, we have recently shown that TNF alpha and interferon-gamma (IFN gamma) act synergistically to block TSH-induced increases in type I 5'D-I activity and 5'D-I gene expression in FRTL-5 rat thyroid cells. Thus, we tested the hypothesis that the activation of one or both pathways is involved in synergistic effect of TNF alpha and IFN gamma on thyroid function. In TSH-stimulated FRTL-5 cells, the addition of 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of PKC, inhibited TSH-induced increases in 5'D-I activity and 5'D-I mRNA levels in a dose-dependent manner. Incubation of FRTL-5 cells with TPA and a minimal effective concentration of IFN gamma (12.5 U/ml) in combination, however, failed to result in a synergistic inhibition of the TSH-induced increase in 5'D-I activity or 5'D-I mRNA levels. Similarly, incubation of FRTL-5 cells with sphingomyelinase (SMase), which converts sphingomyelin to ceramide, inhibited TSH-induced increases in 5'D-I activity and 5'D-I mRNA levels in a dose-dependent manner. Coincubation of FRTL-5 cells with SMase and IFN gamma failed to show a synergistic inhibition of the TSH-induced increase in 5'D-I activity or 5'D-I mRNA levels. Further, incubation of FRTL-5 cells with TPA plus SMase in the presence of IFN gamma failed to result in the synergistic inhibition of TSH-induced increases in 5'D-I activity or 5'D-I mRNA levels. The effect of TPA and SMase on TSH-induced cAMP production was examined. Low concentrations of TPA, which inhibit TSH-induced 5'D-I activity, failed to inhibit TSH-induced cAMP production or the cAMP-induced increase in 5'D-I activity. In contrast, SMase inhibited TSH-induced cAMP production in a dose-dependent manner. In the presence of IFN gamma, however, activation of either or both pathways is not sufficient to result in a synergistic inhibition of 5'D-I activity or 5'D-I gene expression. Together, our results suggest that TNF alpha-induced activation of PKC and hydrolysis of sphingomyelin can inhibit thyroid cell function. The activation of additional signal transduction pathways, however, by TNF alpha is required for the synergistic inhibition of thyroid function by TNF alpha and IFN gamma.