ABSTRACT
Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants, and many are potent carcinogens. Benzo[a]pyrene (B[a]P), one of the best-studied PAHs, is metabolized ultimately to the genotoxin anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE). BPDE triggers stress responses linked to gene expression, cell death and survival. So far, the underlying mechanisms that initiate these signal transduction cascades are unknown. Here we show that BPDE-induced DNA damage is recognized by DNA damage sensor proteins to induce activation of the stress-activated protein kinase (SAPK) p38. Surprisingly, the classical DNA damage response, which involves the kinases ATM and ATR, is not involved in p38-SAPK activation by BPDE. Moreover, the induction of p38-SAPK phosphorylation also occurs in the absence of DNA strand breaks. Instead, increased phosphorylation of p38-SAPK requires the nucleotide excision repair (NER) and DNA damage sensor proteins XPC and mHR23B. Interestingly, other genotoxins such as cisplatin (CDDP), hydrogen peroxide and ultraviolet radiation also enhance XPC-dependent p38-SAPK phosphorylation. In contrast, anti-benzo[c]phenanthrene-3,4-dihydrodiol-1,2-epoxide, the DNA adducts of which are not properly recognized by NER, does not trigger p38-SAPK activation. As a downstream consequence, expression and secretion of the pro-inflammatory cytokine interleukin-6 is induced by BPDE and CDDP in vitro and by CDDP in the murine lung, and depends on XPC. In conclusion, we describe a novel pathway in which DNA damage recognition by NER proteins specifically leads to activation of p38-SAPK to promote inflammatory gene expression.
Subject(s)
Carcinogenesis/metabolism , DNA Adducts/metabolism , DNA Repair/physiology , Interleukin-6/biosynthesis , p38 Mitogen-Activated Protein Kinases/metabolism , 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide/metabolism , 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide/toxicity , Animals , Blotting, Western , Carcinogens/toxicity , Comet Assay , DNA Damage/drug effects , DNA Damage/physiology , DNA-Binding Proteins/metabolism , Enzyme-Linked Immunosorbent Assay , Fibroblasts , HeLa Cells , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mutagens/toxicity , NIH 3T3 Cells , RNA, Small Interfering , Real-Time Polymerase Chain Reaction , Signal Transduction/physiology , TransfectionABSTRACT
A panel of monoclonal antibodies has been produced against alpha-latrotoxin using black widow spider venom. Five of them were characterized relative to their affinity for alpha-latrotoxin and ability to modify the main toxin effects--to increase calcium permeability of synaptosomes, to stimulate the neurotransmitter release and to form the ion channels in artificial lipid membrane. The results reported here show that: (i) the monoclonal antibodies do not alter the alpha-latrotoxin affinity for the membrane acceptor; (ii) two monoclonal antibodies, A6 and A24, can simultaneously inhibit the alpha-latrotoxin induced Ca2+ uptake and GABA release; (iii) monoclonal antibodies A4 completely block the toxin-induced Ca2+ uptake, but decrease partially the rate of GABA release; (iv) monoclonal antibodies A15 that do not modify the alpha-latrotoxin ability to stimulate Ca2+ uptake and GABA release are able to alter the properties of channels formed by the toxin in the artificial lipid bilayer. From these data we hypothesize that the alpha-latrotoxin molecule has separate functional sites which provide a high-affinity binding to the membrane acceptor, the toxin-induced Ca2+ uptake and toxin-stimulated neurotransmitter release. A separate part of alpha-latrotoxin molecule is responsible for the formation of cationic channels in the artificial lipid bilayer.
