Mutagenesis by hydrogen peroxide treatment of mammalian cells: a molecular analysis

Hydrogen peroxide is an oxidizing agent which can be generated intracellularly either during normal metabolism or by treatment with external agents including solar UV radiation. Simian cells (CV-1) transfected with the SV40-based shuttle vector plasmid pZ189 have been treated with H2O2 and then incubated to allow repair and replication of the plasmid. The frequency of mutations at the supF locus of the recovered plasmid increases by a factor of up to four over the spontaneous value. The nucleotide changes associated with 100 spontaneous and 100 H2O2-induced mutants have been determined directly by sequencing a 150 bp fragment that includes the entire supF tRNA coding region. Deletions were observed in ∼45% of both the spontaneous and induced mutants, whereas single or multiple base changes arose in 68 and 57% of the induced and spontaneous mutants respectively. The spectrum of induced mutations is characterized by (i) the occurrence of deletions associated with base changes (16% of all mutants analysed) and (ii) small deletions of 3 bp and less (51% of all deletion mutants sequenced). Sixty-five per cent (15 out of 23) of all small deletions (spontaneous and induced) are associated with runs of between two and five identical bases and eight of them arise at a mutational ‘hotspot' region of five cytosines between bp 172 and 176. The majority (19 out of 30) of completely sequenced deletions observed in the spontaneous spectrum contain either (i) small (2-10 bp) direct repeat sequences that lie immediately outside one deletion terminus and immediately inside the second deletion terminus or (ii) small (2-3 bp) inverted repeat sequences lying immediately inside the two deletion termini. Most deletions that we have observed are therefore likely to arise as a consequence of specific aspects of DNA structur

Gene trap mice reveal an essential function of dual specificity phosphatase Dusp16/MKP-7 in perinatal survival and regulation of Toll-Like Receptor (TLR)-induced cytokine production.

MAPK activity is negatively regulated by members of the dual specificity phosphatase (Dusp) family, which differ in expression, substrate specificity, and subcellular localization. Here, we investigated the function of Dusp16/MKP-7 in the innate immune system. The Dusp16 isoforms A1 and B1 were inducibly expressed in macrophages and dendritic cells following Toll-like receptor stimulation. A gene trap approach was used to generate Dusp16-deficient mice. Homozygous Dusp16tp/tp mice developed without gross abnormalities but died perinatally. Fetal liver cells from Dusp16tp/tp embryos efficiently reconstituted the lymphoid and myeloid compartments with Dusp16-deficient hematopoietic cells. However, GM-CSF-induced proliferation of bone marrow progenitors in vitro was impaired in the absence of Dusp16. In vivo challenge with Escherichia coli LPS triggered higher production of IL-12p40 in mice with a Dusp16-deficient immune system. In vitro, Dusp16-deficient macrophages, but not dendritic cells, selectively overexpressed a subset of TLR-induced genes, including the cytokine IL-12. Dusp16-deficient fibroblasts showed enhanced activation of p38 and JNK MAPKs. In macrophages, pharmacological inhibition and siRNA knockdown of JNK1/2 normalized IL-12p40 secretion. Production of IL-10 and its inhibitory effect on IL-12 production were unaltered in Dusp16tp/tp macrophages. Altogether, the Dusp16 gene trap mouse model identifies an essential role in perinatal survival and reveals selective control of differentiation and cytokine production of myeloid cells by the MAPK phosphatase Dusp16

Selective Expression of the MAPK Phosphatase Dusp9/MKP-4 in Mouse Plasmacytoid Dendritic Cells and Regulation of IFN-beta Production

Item does not contain fulltextPlasmacytoid dendritic cells (pDCs) efficiently produce large amounts of type I IFN in response to TLR7 and TLR9 ligands, whereas conventional DCs (cDCs) predominantly secrete high levels of the cytokines IL-10 and IL-12. The molecular basis underlying this distinct phenotype is not well understood. In this study, we identified the MAPK phosphatase Dusp9/MKP-4 by transcriptome analysis as selectively expressed in pDCs, but not cDCs. We confirmed the constitutive expression of Dusp9 at the protein level in pDCs generated in vitro by culture with Flt3 ligand and ex vivo in sorted splenic pDCs. Dusp9 expression was low in B220(-) bone marrow precursors and was upregulated during pDC differentiation, concomitant with established pDC markers. Higher expression of Dusp9 in pDCs correlated with impaired phosphorylation of the MAPK ERK1/2 upon TLR9 stimulation. Notably, Dusp9 was not expressed at detectable levels in human pDCs, although these displayed similarly impaired activation of ERK1/2 MAPK compared with cDCs. Enforced retroviral expression of Dusp9 in mouse GM-CSF-induced cDCs increased the expression of TLR9-induced IL-12p40 and IFN-beta, but not of IL-10. Conditional deletion of Dusp9 in pDCs was effectively achieved in Dusp9(flox/flox); CD11c-Cre mice at the mRNA and protein levels. However, the lack of Dusp9 in pDC did not restore ERK1/2 activation after TLR9 stimulation and only weakly affected IFN-beta and IL-12p40 production. Taken together, our results suggest that expression of Dusp9 is sufficient to impair ERK1/2 activation and enhance IFN-beta expression. However, despite selective expression in pDCs, Dusp9 is not essential for high-level IFN-beta production by these cells