ABSTRACT
Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta (TGF-beta) superfamily. In the present study, we investigated the effect of BMPs on the production of inducible nitric oxide synthase (iNOS) in the murine macrophage cell line, RAW 264.7, and in mouse peritoneal macrophages. Among the BMPs, only BMP-6 induced iNOS expression in a time-dependent and dose-dependent manner in both cell types. Induction of iNOS was inhibited by both cycloheximide and actinomycin D, indicating that the induction of iNOS expression by BMP-6 requires new protein synthesis. Mechanistic studies revealed that the BMP-6-induced iNOS expression requires both Smads and nuclear factor-kappa B (NF-kappaB) signalling pathways. Furthermore, induction of interleukin-1beta (IL-1beta) was necessary for iNOS induction by BMP-6. These observations suggest that BMP-6 stimulates macrophages to produce iNOS through IL-1beta via Smad and NF-kappaB signalling pathways and that BMP-6 may be an important regulator of macrophages.
Subject(s)
Bone Morphogenetic Protein 6/pharmacology , Macrophages, Peritoneal/drug effects , Nitric Oxide Synthase Type II/biosynthesis , Animals , Antibodies, Neutralizing/immunology , Cells, Cultured , Cycloheximide/pharmacology , Dactinomycin/pharmacology , Humans , Interleukin-1beta/antagonists & inhibitors , Interleukin-1beta/immunology , Interleukin-1beta/metabolism , Macrophages, Peritoneal/enzymology , Mice , NF-kappa B/metabolism , Protein Synthesis Inhibitors/pharmacology , Recombinant Proteins/pharmacology , Signal Transduction , Smad Proteins/metabolism , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/metabolismABSTRACT
PURPOSE AND EXPERIMENTAL DESIGN: Previously, we observed that the activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun NH(2)-terminal kinase (JNK1) is mediated through the activation of apoptosis signal-regulating kinase 1 (ASK1) as a result of the reactive oxygen species-mediated dissociation of glutaredoxin and thioredoxin from ASK1. In this study, we examined whether p38 MAPK and JNK1 are involved in the accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) during ischemia. Human pancreatic cancer MiaPaCa-2 cells were exposed to low glucose (0.1 mmol/L) with hypoxia (0.1% O(2)). RESULTS AND CONCLUSIONS: During ischemia, p38 MAPK and JNK1 were activated in MiaPaCa-2 pancreatic cancer cells. The activated p38 MAPK, but not JNK1, phosphorylated HIF-1alpha. Data from in vivo binding assay of von Hippel-Lindau tumor suppressor protein with HIF-1alpha suggests that the p38-mediated phosphorylation of HIF-1alpha contributed to the inhibition of HIF-1alpha and von Hippel-Lindau tumor suppressor protein interaction during ischemia. SB203580, a specific inhibitor of p38 MAPK, inhibited HIF-1alpha accumulation during ischemia, probably resulting from the ubiquitination and degradation of HIF-1alpha.
Subject(s)
Hypoxia-Inducible Factor 1, alpha Subunit/biosynthesis , Pancreatic Neoplasms/metabolism , Signal Transduction , Adenoviridae/genetics , Apoptosis , Blotting, Western , Catalase/metabolism , Cell Line, Tumor , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , Enzyme Inhibitors/pharmacology , Glucose/metabolism , Glutaredoxins , Humans , Hypoxia/metabolism , Imidazoles/pharmacology , Ischemia , MAP Kinase Kinase Kinase 5/metabolism , Mitogen-Activated Protein Kinase 8/metabolism , Models, Biological , Oxidoreductases/metabolism , Phosphorylation , Plasmids/metabolism , Protein Binding , Pyridines/pharmacology , Reactive Oxygen Species , Thioredoxins/metabolism , Von Hippel-Lindau Tumor Suppressor Protein/metabolism , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Tumor microenvironment, which is characterized by hypoxia, low-glucose concentrations, high-lactate concentrations, low-extracellular pH, can alter the therapeutic response in tumors. In this study, we investigated whether hypoxia affects TRAIL-induced apoptotic death. When human prostate adenocarcinoma DU-145 cells were treated with 50 ng/mL TRAIL or hypoxia for 4 h, the survival was 45.7 and 32.5%, respectively. The combination of TRAIL and hypoxia synergistically increased cell death. Similar results were observed in human prostate adenocarcinoma LNCaP cells. Western blot analysis showed that the hypoxia augmented TRAIL-induced PARP cleavage as well as the activation of caspase-8 and caspase-3, but not caspase-9. Unlike hypoxia, low glucose promoted caspase-9 activation during TRAIL treatment. These results suggest that hypoxia or low glucose-augmented TRAIL cytotoxicity is mediated through the mitochondria-independent pathway or -dependent pathway, respectively.