Nuclear factor-κB2 represses Sp1-mediated transcription at the CD99 promoter

Abstract

Downregulation of the CD99 antigen on the surface of Hodgkin's lymphoma (HL) cells via EBV LMP1-mediated NF-κB suppression of Sp1 transcriptional activity is known to be associated with the appearance of pathogenic Reed-Sternberg cells. Here, we show that in addition, EBV LMP1 heterologous NF-κB activators such as CD30 and CD40 repress the CD99 promoter, which contains multiple Sp1-binding sites but no NF-κB binding sites. In addition, NF-κB-inducing kinase (NIK) repressed the CD99 promoter while NIK kinase mutants and JNK inhibitory protein failed to do so. Of the NF-κB subunits, NF-κB2 (p52) alone or in combination with other Rel subunits consistently inhibited the CD99, while NF-κB1 (p50) showed a marginal repressive effect. Furthermore, while transfection of LMP1 repressed the CD99 promoter in wild-type or NF-κB1 deficient MEFs, the same repression was not observed in NF-κB2 (p52)-deficient MEFs, indicating that NF-κB2 (p52) is required for LMP1-mediated repression of the CD99 promoter. Consistently, basal activity of the CD99 promoter was significantly higher in IKKα(-/-) and IKKβ(-/-) MEFs, but not in IKKΓ(-/-) MEFs compared to the wild-type control MEFs. Sp1-binding sites were directly used in the repression, because a synthetic Sp1 reporter with 10 Sp1-binding sites from the CD99 promoter was repressed by LMP1 or p52 transfection. These data indicate that LMP1-mediated NF-κB2 exhibits the major inhibitory role in the transcription at the CD99 promoter.

Fig. 1.. NF-κB inhibits the Sp1 binding site-rich CD99 promoter (-1643~+123-Luc). (A) Heterologous NF-κB activators, LMP1-CD40 and LMP1-CD30, inhibit the CD99 promoter. (B) Transfection of NIK but not NIK-KM, a kinase mutant defective in kinase activity, represses the CD99 promoter. In (A, B), 293T cells were transfected with the indicated amount of effector, CD99 promoter (-1643 ~+123-Luciferase), and pGKβ-galactosidase, and subjected to reporter assays and Western blotting 24 h after transfection, using equal amounts of protein extracts.

Fig. 2.. LMP1 represses the CD99 promoter via Sp1 sites. (A) 293T cells were co-transfected with LMP1 and a CD99 promoter that has a deletion of Sp1 sites (denoted as black squares). LMP1 and NF- κB2 (p52) repressed any promoter that contained one or more Sp1 site(s). (B) LMP1 represses a synthetic Sp1 reporter (CD99SP 1 × 10Luc), which includes the 10 Sp1 sites collected from the CD99 promoter. (C) In the control, pCDNA3-Sp1 activates the CD99SP 1 × 10Luc reporter, whereas p52 contransfection with the Sp1 represses the same reporter to the basal level 72 h after transient transfection in 293T cells. (D) In B lymphocytes-derived Burkitt’s lymphoma BJAB cells, pcDNA3-Sp1 activates the same CD99SP 1 × 10Luc and -1643~+123-Luc reporter (not shown), while p52 transfection represses the same reporter activity significantly to lower than that of the Sp1 or vector control 24 h after transfection.

Fig. 3.. NF-κB2 (P52) exhibits major inhibitory activity on the CD99 promoter. (A) p52 alone or in combination with RelA, c-Rel, RelB, and p65/c-Rel was transfected into 293T cells for the repression assay. (B) Mouse embryonic fibroblasts from wild-type control mice, and NF-κB2 precursor (p100/p52) - or NF-κB1 precursor (p105/ p50)-deficient mice were transfected with LMP1. (C) MEFs from wild-type control, IKKα^-/-, IKKβ^-/-, and IKKγ^-/- mice were transiently transfected. In (A-C), cells were transiently co-transfected with the -1643~+123-Luc CD99 promoter and pGKβ-galactosidase control reporter, and subjected to reporter assays and Western blotting 20 h (A) or 48 h after transfection, using equal amounts of protein extracts (B). The p52 heteromeric complexes with all other subunits exhibited significant repressive activity in a dose-dependent manner, whereas the p50 heteromeric complexes had no or a much lesser inhibitory effect (Supplementary Fig. 3). (D) LMP1 stimulates p100 processing to p52. Approximately 10⁵ 293T cells were transfected with increasing amounts (0, 0.03, 0.1, 0.3, and 1 μg) of pCDNALMP1, RSV-p52 (0.1 μg), or NIK (0.1 μg) as controls. At posttransfection day 2, cell lysates were separated and transferred to nitrocellulose before being probed with the anti-p52 mouse monoclonal antibody (C-5, Santa Cruz). The processing of p100 by LMP1 results in 3 major bands, likely terminating at aa 445 (not fully processed, upper band) or aa 405 (lower band, p52).