Protein-tyrosine Phosphatase Inhibitors Block Tumor Necrosis Factor-dependent Activation of the Nuclear Transcription Factor NF-kB

Abstract

Most of the inflammatory and proviral effects of tumor necrosis factor (TNF) are mediated through the activation of the nuclear transcription factor NF-kappa B. How TNF activates NF-kappa B, however, is not well understood. We examined the role of protein phosphatases in the TNF-dependent activation of NF-kappa B. Treatment of human myeloid ML-1a cells with TNF rapidly activated (within 30 min) NF-kappa B; this effect was abolished by treating cells with inhibitors of protein-tyrosine phosphatase (PTPase), including phenylarsine oxide (PAO), pervanadate, and diamide. The inhibition was dependent on the dose and occurred whether added before or at the same time as TNF. PAO also inhibited the activation even when added 15 min after the TNF treatment of cells. In contrast to inhibitors of PTPase, okadaic acid and calyculin A, which block serine-threonine phosphatase, had no effect. The effect of PTPase inhibitors was not due to the modulation of TNF receptors. Since both dithiothreitol and dimercaptopropanol reversed the inhibitory effect of PAO, critical sulfhydryl groups in the PTPase must be involved in NF-kappa B activation by TNF. PTPase inhibitors also blocked NF-kappa B activation induced by phorbol ester, ceramide, and interleukin-1 but not that activated by okadaic acid. The degradation of I kappa B protein, a critical step in NF-kappa B activation, was also abolished by the PTPase inhibitors as revealed by immunoblotting. Thus, overall, we demonstrate that PTPase is involved either directly or indirectly in the pathway leading to the activation of NF-kappa B.

The ATM protein is required for sustained activation of NF-κB following DNA damage

“…The nature of the secondary breaks generated after interaction with the DNA replication fork is unknown. Thus either the DNA lesions generated by daunomycin activate NF-kB through another pathway that does not involve ATM (due to di erence in the type of DNA lesions) or, more probably, daunomycin-mediated NFkB activation does even not involve nuclear damage (like UV-mediated NF-kB activation (Devary et al, 1993)) but a cytoplasmic signal, such as ceramide production (Bose et al, 1995;Ja re zou et al, 1996;Singh and Aggarwal, 1995).…”

Overexpression of γ-glutamylcysteine synthetase suppresses tumor necrosis factor-induced apoptosis and activation of nuclear transcription factor-kappa B and activator protein-1

“…To determine the levels of IkBa, postnuclear (cytoplasmic) extracts were prepared (Singh and Aggarwal, 1995) from TNF-treated cells and resolved on 9% SDS-polyacrylamide gels. To determine the levels of NF-kB proteins and p65, nuclear and postnuclear extracts prepared from TNF-treated cells were resolved on 9% SDS ± PAGE.…”

“…To determine the levels of NF-kB proteins and p65, nuclear and postnuclear extracts prepared from TNF-treated cells were resolved on 9% SDS ± PAGE. After electrophoresis, the proteins were electrotransferred to nitrocellulose ®lters, probed with rabbit polyclonal antibodies against IkBa or p65, and detected by chemiluminescence (ECL, Amersham) (Singh and Aggarwal, 1995). The bands obtained were quantitated using Personal Densitometer Scan v1.30 and Image Quant software version 3.3 (Molecular Dynamics).…”

Cellular delivery of a double-stranded oligonucleotide

“…[15][16][17][18] In addition, synthetic double-stranded DNA (dsDNA) with high affinity for NFkB, NFkB decoy, have been shown to block the induction of gene expression mediated by NFkB in vitro 15,[19][20][21][22] and in vivo. 23,24 With the use of decoy oligonucleotides, the function of a particular DNA-binding protein or protein complex can be inhibited since the decoy competes for protein binding with the authentic binding elements, interfering with gene regulation.…”