interleukin-8 and lactacystin

Protein metabolism contributes in the regulation of gut barrier function, which may be altered during inflammatory states. There are three major proteolytic pathways in mammalian cells: lysosomal, Ca(2+)-activated and ubiquitin-proteasome. The regulation of proteolytic activities during inflammation remains unknown in intestine. Intestinal epithelial cells, HCT-8, were stimulated by IL-1beta, IFNgamma and TNFalpha each alone or in combination (Cytomix). Proteolytic activities were assessed using fluorogenic substrates and specific inhibitors, protein expressions by Western blot. Lysosomal and Ca(2+)-activated pathways were not significantly altered by any treatment. In contrast, the activity of ubiquitin-proteasome system was stimulated by IFNgamma and Cytomix (155, 160 versus 100, P<0.05, respectively) but remained unaffected by IL-1beta and TNFalpha. Free ubiquitin expression, but not ubiquitinated proteins, was enhanced by IFNgamma and Cytomix. The expression of proteasome 20S alpha1 subunit, a constitutive proteasome 20S subunit, was not altered, beta5 subunit expression was weakly decreased by Cytomix and inducible beta5i subunit expression was markedly increased in response to IFNgamma and to Cytomix (202, 206 versus 100, P<0.05, respectively). In conclusion, lysosomal, Ca(2+)-activated and constitutive proteasome activities were not affected by IL-1beta, IFNgamma and TNFalpha alone or in combination, in HCT-8 cells. These results suggest that IFNgamma, but not IL-1beta and TNFalpha, increases immunoproteasome, which might contribute to enhanced antigen presentation during inflammatory bowel diseases.

Topics: Acetylcysteine; Blotting, Western; Cell Line, Tumor; Cytokines; Epithelial Cells; Gene Expression; Humans; Hydrolysis; Interferon-gamma; Interleukin-1; Interleukin-8; Intestinal Mucosa; Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Necrosis Factor-alpha; Ubiquitin; Ubiquitin C

Breakdown of cellular proteins is a highly regulated process, and the ubiquitin-proteasome pathway is the major proteolytic system in the cell. It regulates the levels of numerous proteins that control gene expression and cell division, as well as responses to stress and inflammation. Recent studies have reported abnormalities in proteasome function in alcoholic liver disease (ALD). Moreover, a direct relation has been reported between impaired proteasome function and oxidative stress in experimental models of ALD. Neutrophil infiltration is a hallmark of ALD, and activated neutrophils are thought to play a role in the pathology of ALD. As a potent neutrophil chemoattractant and activator, interleukin 8 (IL-8) likely plays a key mechanistic role in many forms of liver injury. In this study, we evaluated the effects of inhibition of proteasome function on expression and release of IL-8 by human fetal hepatocytes and hepatoma cells. Our data demonstrate that inhibition of proteasome function in hepatocytes leads to apoptotic cell death. Decreased hepatocyte survival coincides with enhanced expression of IL-8, both at the protein and the messenger RNA (mRNA) levels. This increase in IL-8 is independent of nuclear factor kappaB (NF-kappaB) activation and is associated with an increase in c-Jun N-terminal kinase (JNK) and activator protein-1 (AP-1) activity. In conclusion, hepatocytes dying because of inhibition of proteasome function produce massive quantities of the proinflammatory chemokine IL-8, possibly resulting in neutrophil infiltration, increased inflammation, and liver injury.

Topics: Acetylcysteine; Apoptosis; Cell Death; Cell Line, Tumor; Chemotaxis, Leukocyte; Cysteine Proteinase Inhibitors; DNA; DNA Fragmentation; Enzyme Activation; Fetus; Hepatocytes; Humans; Interleukin-8; JNK Mitogen-Activated Protein Kinases; Leupeptins; Mitogen-Activated Protein Kinases; Neutrophils; NF-kappa B; Peptide Hydrolases; Proteasome Endopeptidase Complex; RNA, Messenger; Transcription Factor AP-1; Up-Regulation

In this study, we investigated the effects of proteasome inhibibors (MG132 and lactacystin) on interleukin (IL)-8 induction. In human epithelial A549 cells, MG132 and lactacystin induced IL-8 release within the range of 0.1-30 microM. The effect of MG132 resulted from IL-8 gene transcription and was blocked by PD 98059, but was unaffected by GF109203X, Ro 31-8220, or SB 203580. Mutational analysis of the 5' flanking region of the IL-8 gene revealed that activator protein (AP)-1-binding element, but not that element responsive to nuclear factor (NF)-IL-6 or NF-kappaB, was necessary for MG132 stimulation. Consistent with this, MG132 and lactacystin increased the DNA-binding and reporter activities of AP-1, but reduced cytokine-elicited kappaB activation. Moreover, AP-1 stimulation was associated with increased extracellular signal-related kinase (ERK), mitogen-activated protein/ERK kinase (MEK), and c-Jun N-terminal kinase (JNK) phosphorylation, whereas IL-8 activity was sensitive to the dominant-negative mutants of JNK1, JNK2, SEK, ASK, ERK2, and Ras, but not those of MEKK1, TAK, and p38 mitogen-activated protein kinase. In addition, activations of the IL-8 gene and AP-1 by MG132 and lactacystin were inhibited by GSH and NAC. Herein we present a novel action of proteasome inhibitors, possibly through ROS production, of targeting the upstream signaling molecules, ERK and JNK, which leads to AP-1 activation and IL-8 gene expression.

Topics: Acetylcysteine; Cell Line; Chemotaxis; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Enzyme Activation; Epithelial Cells; Genes, Reporter; Humans; Interleukin-8; JNK Mitogen-Activated Protein Kinases; Leupeptins; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Multienzyme Complexes; Neutrophils; NF-kappa B; Proteasome Endopeptidase Complex; ras Proteins; Reactive Oxygen Species; Transcription Factor AP-1; Transcription Factors; Tumor Cells, Cultured

In this report we explored the effects of proteasome inhibitors (MG132, aLLN, lactacystin and MG262) on interleukin-8 (IL-8) induction. In HEK293 cells, proteasome inhibitors could concentration-dependently increase IL-8 promoter and activator protein-1 (AP-1) activities, but inhibited nuclear factor (NF)-kappa B activation induced by cytokines. The stimulating effects on IL-8 promoter and AP-1 were reduced by N-acetylcysteine, glutathione, diphenyleneiodonium, rotenone and antimycin A. Fluorescent analysis using 2',7'-dichlorodihydrofluorescin diacetate further confirmed the abilities of proteasome inhibitors to induce reactive oxygen species (ROS) production. These results suggest that ROS production by proteasome inhibitors leads to AP-1 activation, which in the absence of NF-kappa B activation still transactivates IL-8 gene expression.

Topics: Acetylcysteine; Antioxidants; Cell Line; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Gene Expression Regulation; Genes, Reporter; Humans; Interleukin-8; Leupeptins; Luciferases; Multienzyme Complexes; Promoter Regions, Genetic; Proteasome Endopeptidase Complex; Reactive Oxygen Species; Recombinant Proteins; Transcription Factor AP-1; Transfection