Induction of the RelB NF-kappaB subunit by the cytomegalovirus IE1 protein is mediated via Jun kinase and c-Jun/Fra-2 AP-1 complexes

Abstract

We recently demonstrated that the cytomegalovirus (CMV) immediate-early 1 (IE1) protein induces transcription of the gene encoding the RelB NF-kappaB subunit. The mechanism of this activation has been explored here. We report that the induction of the relB promoter by IE1 protein is mediated via activation of JNK and AP-1. The region controlling relB promoter induction was mapped to the upstream approximately 600-bp region between -1694 and -1096 bp. IE1 stimulated AP-1 activity in NIH 3T3 cells. Competition electrophoretic mobility shift assay (EMSA) confirmed the presence of one bona fide AP-1 element centered at -1503 bp. Introduction of a G-to-C mutation in the AP-1 binding site within the distal region of the relB promoter eliminated its activation by IE1 in both NIH 3T3 fibroblasts and vascular smooth muscle cells (SMCs). Supershift EMSA identified c-Jun, Fra-2, and c-Fos in AP-1 binding complexes in IE1 transfected NIH 3T3 cells. IE1 induced c-Jun phosphorylation, and treatment with SP600125, a selective JNK inhibitor, as well as overexpression of JNK-binding domain of JIP1, blocked IE1-mediated induction of AP-1 and relB promoter activity in NIH 3T3 cells and SMCs. Ectopic expression of c-Jun plus Fra-2, but not c-Fos, induced relB promoter activity. The relB promoter has two proximal NF-kappaB elements, and c-Jun/Fra-2 worked in synergy with p50/p65 NF-kappaB complexes. Overall, these findings demonstrate for the first time the role of AP-1 in transcriptional regulation of a gene encoding an NF-kappaB subunit, and its involvement in induction of RelB activity by the CMV IE1 protein.

FIG. 1.

The induction of the relB promoter by IE1 requires the distal region of the promoter. (A) Structures of the p1.7, p1.1, and p0.6 relB promoter luciferase reporter vectors. The positions of the NF-κB elements and putative AP-1 sites are indicated. (B) NIH 3T3 cells were transiently transfected, in triplicate, with the indicated dose of pON2205 vector expressing IE1 protein; 0.5 μg of either p1.7, p1.1, or p0.6 relB promoter-Luc vector; 0.5 μg of SV40 β-Gal expression vector; and pBluescript for a total of 3 μg of DNA. Values of luciferase normalized to β-Gal activity (Relative luciferase units) are presented ± the standard deviation. Untreated p1.7, p1.1, and p0.6 relB promoter-Luc activities were set at 1.

FIG. 2.

IE1 induces activity of 4XTRE-Luc, four-copy AP-1 element-driven reporter construct, in NIH 3T3 cells. NIH 3T3 cells were transiently transfected, in triplicate, with the indicated dose of pON2205 vector expressing IE1 protein, 0.5 μg of 4XTRE-Luc vector, 0.5 μg of SV40 β-Gal expression vector, and pBluescript for a total of 3 μg of DNA. Relative luciferase units are presented ± the standard deviation. Activity of the control 4XTRE-Luc vector DNA was set at 1.

FIG. 3.

The distal region of the relB promoter contains an AP-1 binding site. (A) NIH 3T3 cell nuclear extracts (5 μg) were subjected to competition EMSA by using the oligonucleotide containing the consensus AP-1 element as a probe and 10- or 30-fold excess unlabeled oligonucleotides containing either the consensus (Con) AP-1 element or the putative AP-1 element Puta-AP-1(1) or Puta-AP-1(2), as indicated. (B) NIH 3T3 cell nuclear extracts (5 μg) were subjected to competition EMSA by using either the consensus AP-1 element, Puta-AP-1(1), or Puta-AP-1(2) as a probe, and 10- or 30-fold excess unlabeled consensus AP-1 oligonucleotide. ns, Position of non-AP-1 band.

FIG. 4.

Mutation of the AP-1 element blocks AP-1 binding and reduces RelB induction by IE1. (A) NIH 3T3 cells nuclear extracts (5 μg) were subjected to competition EMSA with labeled relB promoter WT AP-1 element as probe, and excess unlabeled oligonucleotides containing either a WT or mutant (Mut) relB promoter AP-1 site. (B and C) NIH 3T3 cells or SMCs at 50 to 70% confluence were transiently transfected, in triplicate, with the indicated dose of pON2205 vector expressing IE1 protein, 0.5 μg of WT p1.7 or AP-1 Mut-p1.7 relB promoter-Luc, 0.5 μg of SV40 β-Gal expression vector and pBluescript for a total of 3 μg of DNA. Relative luciferase units are presented ± the standard deviation. Untreated WT and AP-1 Mut-p1.7 relB promoter-Luc activities were set at 1 in NIH 3T3 cells (B) or SMCs (C).

FIG. 5.

c-Jun, Fra-2, and c-Fos bind to the distal AP-1 element of the relB promoter. Nuclear extracts were isolated from NIH 3T3 cells 48 h after transfection with 10 μg of pON2205 vector expressing IE1 protein, and samples were subjected to EMSA in the absence (−) or presence of the indicated antibody using as a probe oligonucleotides containing either the AP-1 element upstream of the relB promoter (A) or the consensus AP-1 (B). Alternatively, in the right panels, antibodies were added to the oligonucleotides, as indicated, in the absence of extracts to verify the specificity of the supershifted bands. ns, Nonspecific band.

FIG. 6.

CMV IE1 protein activates c-Jun phosphorylation. NIH 3T3 cells were transfected with 10 μg of pON2205 vector expressing IE1 protein (IE1) or pCMV empty vector DNA (EV). After 4 h, cells were treated with DMSO, 20 μM SP600125, or 40 μM SP600125 for 20 h. Total cell lysates were prepared, and samples (30 μg) were subjected to immunoblot analysis for IE1, c-Jun, phospho-Ser63 c-Jun (P-c-Jun), c-Fos, Fra-2, JunB, JunD, and β-actin (as a loading control).

FIG. 7.

SP600125, the selective inhibitor of the JNK signaling pathway, blocks relB promoter induction by IE1. NIH 3T3 cells were transiently transfected, in triplicate, with the indicated dose of pON2205 vector expressing IE1 protein, and either 0.5 μg of p1.7 relB promoter-Luc (A) or 4XTRE-Luc (B), 0.5 μg of SV40 β-Gal expression vector, and pBluescript for a total of 3 μg of DNA. After 4 h, cells were treated with carrier DMSO (control) or 30 μM SP600125. Cells were harvested after 20 h, the luciferase and β-Gal activities were measured, and normalized values were obtained. Relative (Rel.) luciferase units are presented ± the standard deviation. Control p1.7 relB promoter-Luc activity (A) and untreated 4XTRE-Luc activity (B) were set at 1. (C) NIH 3T3 cells were transiently transfected with 10 μg of pON2205 vector expressing IE1 protein (IE1) or pCMV empty vector DNA (EV). After 4 h, cells were treated with DMSO or 30 μM SP600125 for 20 h, the RNA was isolated, and samples (20 μg) were subjected to Northern blot analysis for relB mRNA (upper panel). Equal loading was confirmed by GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA level (lower panel).

FIG. 8.

Inhibition of the JNK pathway blocks IE1-mediated activation of the relB promoter. (A) 4XTRE-Luc. NIH 3T3 cells were transiently transfected, in triplicate, with 1 μg of pCMV empty vector or pON2205 vector expressing IE1 protein; 0, 1, or 2 μg of pCMV FJIP1; 0.25 μg of 4XTRE-Luc; 0.25 μg of SV40 β-Gal expression vector; and pBluescript for a total of 3.5 μg of DNA. After 24 h, cells were harvested, and the luciferase activity and β-Gal activity were measured. Relative luciferase units are presented ± the standard deviation. The control, empty vector DNA sample was set at 1. For the lower part of panel A, samples of cell lysates (20 μg) from reporter transfection analyses were subjected to immunoblot analysis for IE1 and β-actin. (B) p1.7 relB promoter. NIH 3T3 cells were transiently transfected, in triplicate, with the 1 μg of pCMV empty vector or pON2205 vector expressing IE1 protein, the indicated dose of pCMV FJIP1, 0.25 μg of p1.7 relB promoter-Luc, 0.25 μg of SV40 β-Gal expression vector, and pBluescript for a total of 3.5 μg of DNA. After 24 h, samples were processed, and data are presented as described above. (C) SMCs were transiently transfected, in triplicate, with 0 or 0.2 μg of pON2205 vector expressing IE1 protein, the indicated dose of pCMV FJIP1, 0.5 μg of p1.7 relB promoter-Luc, 0.5 μg of SV40 β-Gal expression vector, and pBluescript for a total of 2 μg of DNA. After 24 h, samples were processed, and data are presented as described above.

FIG. 8.

Inhibition of the JNK pathway blocks IE1-mediated activation of the relB promoter. (A) 4XTRE-Luc. NIH 3T3 cells were transiently transfected, in triplicate, with 1 μg of pCMV empty vector or pON2205 vector expressing IE1 protein; 0, 1, or 2 μg of pCMV FJIP1; 0.25 μg of 4XTRE-Luc; 0.25 μg of SV40 β-Gal expression vector; and pBluescript for a total of 3.5 μg of DNA. After 24 h, cells were harvested, and the luciferase activity and β-Gal activity were measured. Relative luciferase units are presented ± the standard deviation. The control, empty vector DNA sample was set at 1. For the lower part of panel A, samples of cell lysates (20 μg) from reporter transfection analyses were subjected to immunoblot analysis for IE1 and β-actin. (B) p1.7 relB promoter. NIH 3T3 cells were transiently transfected, in triplicate, with the 1 μg of pCMV empty vector or pON2205 vector expressing IE1 protein, the indicated dose of pCMV FJIP1, 0.25 μg of p1.7 relB promoter-Luc, 0.25 μg of SV40 β-Gal expression vector, and pBluescript for a total of 3.5 μg of DNA. After 24 h, samples were processed, and data are presented as described above. (C) SMCs were transiently transfected, in triplicate, with 0 or 0.2 μg of pON2205 vector expressing IE1 protein, the indicated dose of pCMV FJIP1, 0.5 μg of p1.7 relB promoter-Luc, 0.5 μg of SV40 β-Gal expression vector, and pBluescript for a total of 2 μg of DNA. After 24 h, samples were processed, and data are presented as described above.

FIG. 9.

Expression of c-Jun and Fra-2 induces the relB promoter. NIH 3T3 cells were transiently transfected, in triplicate, with 0.5 μg of the indicated AP-1 subunit expression vector, 0.5 μg of WT p1.7 or AP-1 Mut-relB promoter-Luc, 0.5 μg of SV40 β-Gal expression vector, and pCI empty vector for a total of 3 μg of DNA. (Inset) NIH 3T3 cells were transiently transfected, in triplicate, with 0.5 μg of the indicated AP-1 subunit expression vector, 0.5 μg of 4XTRE-Luc vector, 0.5 μg of SV40 β-Gal expression vector, and pCI empty vector for a total of 3 μg of DNA. Values of luciferase normalized to β-Gal activity (in relative luciferase units) are presented ± the standard deviation. Control p1.7 relB promoter- and 4XTRE-Luc activities were set at 1.

FIG. 10.

Synergy between c-Jun/Fra-2 AP-1 complexes and p50/p65 NF-κB complexes potently activate the relB promoter. (A) Scheme of the structures of the WT and NF-κB/AP-1 mutant p1.7 relB promoter luciferase reporter vectors. Mutant elements are indicated by an X. (B) NIH 3T3 cells were transiently transfected, in triplicate, with 0.25 μg of the indicated AP-1 or NF-κB subunit expression vector, 0.5 μg of WT or the indicated NF-κB/AP-1 mutant p1.7 relB promoter-Luc, 0.5 μg of SV40 β-Gal expression vector, and pCI empty vector for a total of 3 μg of DNA. Values of luciferase normalized to β-Gal activity (in relative luciferase units) are presented ± the standard deviation. Control p1.7 relB promoter-Luc activities were set at 1. (Inset) NIH 3T3 cells were transiently transfected with 5 μg of EV and c-Jun/Fra-2 alone or in combination with 5 μg of p50/RelA expression vectors. After 48 h, the RNA was harvested, and samples (20 μg) were subjected to Northern blot analysis for relB mRNA or GAPDH as a control for equal loading.