Induction of cyclooxygenase-2 by Epstein-Barr virus latent membrane protein 1 is involved in vascular endothelial growth factor production in nasopharyngeal carcinoma cells - PubMed (original) (raw)

Induction of cyclooxygenase-2 by Epstein-Barr virus latent membrane protein 1 is involved in vascular endothelial growth factor production in nasopharyngeal carcinoma cells

S Murono et al. Proc Natl Acad Sci U S A. 2001.

Abstract

Cyclooxygenase-2 (COX-2) is an inducible form of COX and is overexpressed in diverse tumors, raising the possibility of a role for COX-2 in carcinogenesis. In addition, COX-2 contributes to angiogenesis. The Epstein-Barr virus (EBV) oncoprotein, latent membrane protein 1 (LMP1), is detected in at least 70% of nasopharyngeal carcinoma (NPC) and all EBV-infected preinvasive nasopharyngeal lesions. We found that in specimens of LMP1-positive NPC, COX-2 is frequently expressed, whereas LMP1-negative NPC rarely express the enzyme. We next found that expression of LMP1 in EBV-negative nasopharyngeal epithelial cells induced COX-2 expression. Coexpression of IkappaBalpha(S32A/S36A), which is not phosphorylated and prevents NF-kappaB activation, with LMP1 showed that NF-kappaB is essential for induction of COX-2 by LMP1. We also demonstrate that NF-kappaB is involved in LMP1-induced cox-2 promoter activity with the use of reporter assays. Two major regions of LMP1, designated CTAR1 and CTAR2, are signal-transducing domains of LMP1. Constructs expressing either CTAR1 or CTAR2 induce COX-2 but to a lesser extent than wild-type LMP1, consistent with the ability of both regions to activate NF-kappaB. Furthermore, we demonstrate that LMP1-induced COX-2 is functional because LMP1 increased production of prostaglandin E(2) in a COX-2-dependent manner. Finally, we demonstrate that LMP1 increased production of vascular endothelial growth factor (VEGF). Treatment of LMP1-expressing cells with the COX-2-specific inhibitor (NS-398) dramatically decreased production of VEGF, suggesting that LMP1-induced VEGF production is mediated, at least in part, by COX-2. These results suggest that COX-2 induction by LMP1 may play a role in angiogenesis in NPC.

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Figures

Figure 1

Expression of COX-2 and LMP1 in NPC (A) and induction of COX-2 by LMP1 in nasopharyngeal epithelial cells (B and_C_). (A) Lysates obtained from each tumor as indicated were analyzed for COX-2 and LMP1 expression by Western blotting. (B) 422F6 is an EBV-negative clone derived from the EBV-positive NPC-KT cell line. Two different amounts of LMP1 expression plasmid were transiently transfected into 422F6 cells and analyzed by Western blotting. Ad-AH cells treated with PMA (100 ng/ml) were used as a positive control for COX-2 induction. (C) Ad-AH cells stably expressing either pcDNA3 or LMP1 were analyzed.

Figure 2

NF-κB is involved and essential for COX-2 induction by LMP1. Suppression of LMP1-induced COX-2 expression by IκBα(S32A/S36A) is shown. COX-2 expression was analyzed with or without coexpression of IκBα(S32A/S36A) (indicated as srIκB) with LMP1 in 422F6 cells (A) or in Ad-AH cells (B) by Western blotting. (C) Induction of nuclear factor binding to NF-κB binding sequence in the cox-2 promoter by LMP1 and its suppression by IκBα. Nuclear extracts of 293 cells transfected as indicated were mixed with 32P-labeled NF-κB probe and analyzed by electrophoretic mobility shift assay. An excess of nonlabeled NF-κB (indicated as κB) or NF-IL6 (indicated as IL6) probe (100×) was used as competitor (NF-κB, 5′-CAGGAGAGT

GGGGACTACC

CCCTCTGCT-3′; NF-IL6, 5′-CACCGGGC

TTACGCAAT

TTTTTTAA-3′). Underlined letters indicate binding sequences in the promoter of the cox-2 gene. srIκB, IκBα(S32A/S36A); NS, nonspecific binding.

Figure 3

Both CTAR1 and CTAR2 of LMP1 partially induce COX-2 through NF-κB. (A) COX-2 expression in each LMP1 mutant-expressing 422F6 cells as indicated was analyzed by Western blotting. Involvement of NF-κB in COX-2 induction by each CTAR was analyzed by coexpression of IκBα(S32A/S36A) (indicated as srIκB) with LMP1 1–231 (B) or LMP1 Δ187–351 (C) in 422F6 cells.

Figure 4

LMP1 induces cox-2 promoter activity. (A) Schematic diagram of cox-2 promoter reporter constructs. X indicates the mutation in the NF-κB binding site in the_cox-2_ promoter region. (B) Relative luciferase activity with each construct from three independent duplicate experiments is shown with the standard deviation.

Figure 5

LMP1 increases production of PGE2 through induction of COX-2. (A) Conditioned medium was diluted, and production of PGE2 was analyzed with EIA. PMA, 100 ng/ml PMA; NS-398, 20 μM NS-398. (B) One-hundred micrograms of lysates from matched samples used in A were analyzed for expression of COX-2 and LMP1 by Western blotting.

Figure 6

LMP1 increases VEGF production, at least in part, in a COX-2-dependent manner. (A) The same conditioned media used in Fig. 5 were analyzed for VEGF production by Western blotting. PMA, 100 ng/ml PMA; NS-398, 20 μM NS-398. (B) Enzymatic activity of MMP-2 and MMP-9 in conditioned media was analyzed by gelatin zymography. PMA, 100 ng/ml; NS-398, 20 μM.

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Induction of cyclooxygenase-2 by Epstein-Barr virus latent membrane protein 1 is involved in vascular endothelial growth factor production in nasopharyngeal carcinoma cells - PubMed (original) (raw)