Tyrosine Phosphatase-Dependent/Tyrosine Kinase-Independent Induction of Nuclear Factor-κB by Tumor Necrosis Factor-α: Effects on Prostaglandin Endoperoxide Synthase-2 mRNA Accumulation
Abstract
We previously have demonstrated that tumor necrosis factor-α (TNF-α) increases prostaglandin endoperoxide synthase-2 (PGHS-2) mRNA accumulation and tyrosine phosphorylation in the fibrosarcoma cell line, MCA-101. Tyrosine kinase inhibitor, genistein, and tyrosine phosphatase inhibitor, phenylarsine oxide (PAO), blocked TNF-α-mediated induction of PGHS-2 mRNA in these cells. Because the PGHS-2 promoter has a nuclear factor-κB (NF-κB) binding motif, which is important for PGHS-2 gene transcription in some cell types, we have evaluated the effects of tyrosine kinase inhibitors and PAO on TNF-α-induced NF-κB activation. TNF-α (1 nM) rapidly induced translocation of NF-κB, an event accompanied by degradation of inhibitory protein IκB-α. N-tosyl-l-phenylalanine chloromethyl ketone (TPCK), a serine protease inhibitor, inhibited IκB-α degradation and NF-κB activation in response to TNF-α in a dose-dependent manner (25, 50, 100 μM). TPCK also inhibited PGHS-2 mRNA accumulation. These data suggest that NF-κB contributed to PGHS-2 mRNA accumulation in MCA-101 cells stimulated with TNF-α. PAO (2.4 μM) completely abolished activation of NF-κB and degradation of IκB-α induced by TNF-α at a concentration that blocked PGHS-2 mRNA accumulation. However, four tyrosine kinase inhibitors, genistein, tyrphostin 47, herbimycin A and erbstatin, failed to block translocation of NF-κB and degradation of IκB-α. These data demonstrate that tyrosine kinase pathways are not required for TNF-α-induced NF-κB activation in MCA-101 cells and suggest that signaling via these pathways mediates TNF-α-induced PGHS-2 mRNA accumulation via an NF-κB-independent mechanism. Moreover, an upstream tyrosine phosphatase pathway may mediate PGHS-2 mRNA accumulation by TNF-α via an NF-κB-dependent mechanism.
Footnotes
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Send reprint requests to: Dr. Keyvan Mahboubi, Department of Pharmacology, New York Medical College, Valhalla, NY 10595.
- Abbreviations:
- PGHS-2
- prostaglandin endoperoxide synthase-2
- NF-κB
- nuclear factor-κB
- MCA
- methylcholanthrene
- TNF-α
- tumor necrosis factor-α
- TBST
- tris buffer saline tween
- SDS
- sodium dodecyl sulfate
- PBS
- phosphate-buffered saline
- EMSA
- electrophoresis mobility shift assay
- C/EBPβ
- CCAAT/enhancer binding protein β
- CRE
- cyclic AMP response element
- PMSF
- phenylmethylsulfonyl fluoride
- PAO
- phenylarsine oxide
- PTKs
- protein tyrosine kinases
- PTPase(s)
- protein tyrosine phosphatases
- TPCK
- N-tosyl-l-phenylalanine chloromethyl ketone
- MTT
- 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium
- PAGE
- polyacrylamide gel electrophoresis
- HEPES
- N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid
- EDTA
- ethylenediaminetetraacetic acid
- DTT
- dithiothreitol
- SSC
- standard saline citrate
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- Received April 8, 1997.
- Accepted January 27, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
