Sulfiredoxin (Srx) is an enzyme that catalyzes the reduction of Pamidronate Disodium cysteine sulfinic acid of hyperoxidized peroxiredoxins and exerts a protective antioxidant role. mutants were used as templates to generate double Pamidronate Disodium or triple mutants in which two or three AP-1 sites were mutated respectively. FIGURE 2. Nucleotide sequence of the mouse Srx promoter made up of potential AP-1 and NF-κB sites as well as ARE. Nucleotides are numbered relative to the transcription start site (+1) shown in enzyme and was then expressed as -fold increase relative to the normalized value for control cells. Chromatin Immunoprecipitation RAW264.7 cells produced in 15-cm dishes were stimulated by LPS (100 ng/ml) for 1 h washed with 1× phosphate-buffered saline (PBS) and fixed by adding 27 ml of 1× PBS made up of 1% formaldehyde. The dishes were rocked for 10 min at room temperature and the cross-linking reaction was stopped by adding 3 ml of 1 1.25 m glycine (final 0.125 m) and rocking for 5 min. The cells were washed twice with ice-cold 1× PBS scraped in 1× PBS made up of protease inhibitors and harvested. The cells were resuspended in 3 ml of lysis buffer (1× PBS 1 Nonidet P-40 0.5% sodium deoxycholate 0.1% SDS 1 mm EDTA and protease inhibitors) and then sonicated using a Branson digital sonifier on power setting 25% for 40 rounds of 1 1 s; all samples were kept on ice at all times. Following sonication a portion of the sonicated answer was uncross-linked for analysis of proper shearing of genomic DNA. The extracts were clarified by Pamidronate Disodium centrifuge at 10 0 rpm for 15 min at 4 °C and were aliquoted for the immunoprecipitation. One Pamidronate Disodium aliquot was set aside to serve as an input control. Other aliquots were incubated with antibodies specific for c-Jun and c-Fos or normal rabbit IgG overnight with rotation. Immune complexes were precipitated by incubating with the salmon sperm DNA/protein A-agarose for 1 h at 4 °C with rotation. The resins were washed with lysis buffer three times and resuspended in elution buffer (1% SDS and 0.1 m NaHCO3). The immunoprecipitated samples were eluted from the resins by Rabbit Polyclonal to MEKKK 4. shaking for 15 min and were incubated at 65 °C for 4 h to reverse the formaldehyde cross-links. The resulting DNA sample was incubated with proteinase K (0.1 mg/ml) in the buffer containing 40 mm Tris-HCl (pH 8.0) and 10 mm EDTA at 50 °C for 90 min and was subsequently purified with QIAEX II resin (Qiagen). The immunoprecipitated DNA was quantified by performing PCR with primers (5′-GAG GGC CTG AGT CAC CAC-3′ and 5′-CTG ACC TAG CTG CCC ACT G-3′). RESULTS Transcriptional Induction of Srx Gene by LPS in Mouse BMM and RAW264.7 Cells Expression of the Srx gene by LPS was investigated in RAW264.7 macrophage cells. Srx protein was considerably induced by LPS (Fig. 1synthesis or actinomycin D which inhibits cellular transcription. LPS-mediated Srx induction was blocked by pretreatment with both cycloheximide and actinomycin D suggesting that LPS-mediated Srx induction was regulated around the transcriptional level (Fig. 1and and and and and (40) showed that both proximal and distal AP-1 sites are important for tumor promoter-induced Srx promoter activity they did not pay attention to the central AP-1 site. Also two major components of AP-1 c-Jun and c-Fos were induced and recruited to the AP-1 site of the Srx promoter in response to LPS. These results suggest that LPS-mediated Srx induction requires AP-1. The consensus sequence TGAGTCA recognized by AP-1 is usually often embedded within AREs (47). It was exhibited that LPS stimulation of human monocytes induces the expression of NQO1 and HO-1 which are regulated by Nrf2 (33 34 In this study Nrf2 was induced and activated in mouse macrophages stimulated with LPS. Given that the proximal AP-1 site of the Srx promoter is also embedded within AREs and that its first three nucleotides TGA correspond to the core and essential nucleotides of AREs positioning in forward and reverse directions respectively (see Fig. 2) mutation of these nucleotides within the proximal AP-1 site (TGAGTCA → CAGGTCA; the changed nucleotides are shown in boldface type) might lead to inactivation of AREs as well as the AP-1 site. Mutation of the proximal AP-1 site resulted in a partial decrease of the LPS-induced promoter activity suggesting that ARE is usually in part involved in LPS-mediated Srx induction. In Nrf2-deficient macrophages however the mRNA level of Srx was never induced by LPS treatment like NQO1 a target of Nrf2. This discrepancy is probably caused by two possibilities. One is incomplete inactivation of AREs by mutation of the.