Regulation of B7.1 Costimulatory Molecule Is Mediated by the IFN Regulatory Factor-7 through the Activation of JNK in Lipopolysaccharide-Stimulated Human Monocytic Cells

Abstract

The engagement of CD28 or CTLA-4 with B7.1 provides the essential second costimulatory signal that regulates the development of immune responses, including T cell activation, differentiation, and induction of peripheral tolerance. The signaling molecules and the transcription factors involved in B7.1 regulation are poorly understood. In this study we investigated the role of MAPKs in the regulation of LPS-induced B7.1 expression in human monocytes and the promonocytic THP-1 cells. Our results show that LPS-induced B7.1 expression in monocytic cells did not involve the activation of either p38 or ERKs. Using the JNK-specific inhibitor SP600125, small interfering RNAs specific for JNK1 and JNK2, and agents such as dexamethasone that inhibit JNK activation, we determined that LPS-induced B7.1 expression was regulated by JNK MAPK in both monocytes and THP-1 cells. In addition, we identified a distinct B7.1-responsive element corresponding to the IFN regulatory factor-7 (IRF-7) binding site in the B7.1 promoter responsible for the regulation of LPS-induced B7.1 transcription. Furthermore, SP600125 and dexamethasone inhibited LPS-induced IRF-7 activity. Taken together, these results suggest that LPS-induced B7.1 transcription in human monocytic cells may be regulated by JNK-mediated activation of the IRF-7 transcription factor.

Activation of JNK-dependent Pathway Is Required for HIV Viral Protein R-induced Apoptosis in Human Monocytic Cells

“…Western Blot Analysis-Cells were treated with the indicated concentration of inhibitors for 2 h followed by stimulation with Vpr peptide(s) for various times for analysis of MAPK activation and Bcl2/c-IAP1 expression by Western immunoblotting as described earlier (22,28). Briefly total proteins were subjected to SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Bio-Rad).…”

“…Blood was obtained from healthy volunteers after approval of the protocol by the ethics review committee of the Children's Hospital of Eastern Ontario, Ottawa, Canada. Peripheral blood mononuclear cells were isolated by density gradient centrifugation over Ficoll-Hypaque (GE Healthcare) as described previously (22). The cell layer consisting mainly of mononuclear cells was collected and washed twice in phosphate-buffered saline containing 2% EDTA followed by incubation with autoMACS CD14 microbeads (Miltenyi Biotech Inc., Auburn, CA) for 15 min at 4°C.…”

“…Total RNA (1 g) was reverse transcribed using Moloney murine leukemia virus reverse transcriptase (PerkinElmer Life Sciences) as described previously (22,28). Equal aliquots (5 l) of cDNA were subsequently amplified for Bcl2, c-IAP1, and ␤-actin.…”

“…The amino acid sequence of these peptides is as follows: Vpr-(52-96), DTWAGVEAIIRILQQ-LLFIHFRIGCRHSR IGVTRQRRARNGASRS; Vpr-(1-45), MEQAPEDQGPQREPYNEWTLELLEELKSEAVRHFPRIW-LHNLGQH. Because of the high propensity of Vpr peptides to bind to proteins, cells (1 ϫ 10 6 /ml) were treated with peptides in an isotonic buffer (13 mM HEPES, 2.4% glucose, 68 mM NaCl, 1.3 mM KCL, 4 mM Na 2 HPO 4 , 0.7 mM KH 2 PO 4 , pH 7.2) for 30 min followed by addition of culture medium as described previously (27).Western Blot Analysis-Cells were treated with the indicated concentration of inhibitors for 2 h followed by stimulation with Vpr peptide(s) for various times for analysis of MAPK activation and Bcl2/c-IAP1 expression by Western immunoblotting as described earlier (22,28). Briefly total proteins were subjected to SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Bio-Rad).…”

“…Briefly a 50 pM concentration of either stealth JNK or control RNA was incubated for 45 min at room temperature with 3 l of FuGENE 6 in 100 l of serum-free IMDM to allow formation of RNA-liposome complexes. These complexes were then added to the cell suspension (10 6 /ml) for 24 h followed by treatment with Vpr peptide(s) for either 2 or 24 h after which cells were analyzed for JNK phosphorylation by Western blot analysis or for apoptosis, cell cycle arrest, and mitochondrial membrane potential, respectively.Electrophoretic Mobility Shift Assays-Electrophoretic mobility shift assays were performed as described earlier (22,28). Briefly cells were treated with Vpr peptide(s) for 3 h in the presence or the absence of indicated inhibitors.…”