Tax 1-independent induction of vascular endothelial growth factor in adult T-cell leukemia caused by human T-cell leukemia virus type 1

Abstract

Adult T-cell leukemia (ATL) is caused by human T-cell leukemia virus type 1 (HTLV-1). Elevated expression of vascular endothelial growth factor (VEGF) in ATL patients is associated with leukemic cell invasion and infiltration in different organs. The regulatory protein Tax 1 encoded by HTLV-1 plays a pivotal role in T-cell transformation by deregulating the function and expression of several cellular factors. In the present study, we examined the effect of Tax 1 on VEGF expression at transcriptional and posttranscriptional levels in order to elucidate the regulatory mechanisms involved. Using functional assays, we demonstrate that Tax 1 downregulates the VEGF promoter through a cluster of Sp1 sites located close to the transcriptional start site. Using gel mobility shift assays, we show that Tax 1 reduced Sp1:DNA complex formation. We demonstrate that the level of secreted VEGF was significantly lower in Tax 1-transfected 293T cells compared to nontransfected cells, which is consistent with the observed downregulatory effect of Tax 1 at the transcription level. We showed that VEGF was secreted by HTLV-1-transformed and nontransformed cells, irrespective of Tax 1 expression. Overall our data indicate that, contrary to a previous report, Tax 1 downregulates VEGF expression and suggest there are Tax 1-independent mechanisms of VEGF activation in ATL.

FIG. 1.

Schematic representation of VEGF full-length and deleted promoter PGL3 constructs showing the relative positions of binding sites for hypoxia-inducible factor 1 (HIF-1), AP-1, and Sp1.

FIG. 2.

Tax 1 downregulates VEGF promoter activity. (a) Luciferase assays were carried out on lysates from 293T cells transfected with the full-length VEGF promoter construct PGL3-2.6 and incubated under normoxic and hypoxic conditions. The lower panel shows the expression level of HIF-1α in both conditions as determined by Western blotting. 293T (b), COS 7 (c), and HeLa (d) cells were cotransfected with PGL3-2.6 and the indicated concentrations of pCA-Tax1-HIS or empty pCA vector together with pRLTK, which was used as an internal control. Luciferase activity was quantified after 48 h and was normalized to thymidine kinase (TK) values. Basal activity was set at 100%. Differences in normalized luciferase activity between transfected and nontransfected cells were significant as determined by Student's t test. The lower panels in each figure show the levels of Tax 1 expression in lysates used for luciferase assays as determined by Western blotting.

FIG. 3.

The Sp1 sites, located between positions −194 and −51, are involved in Tax 1-mediated downregulation of the VEGF promoter. 293T (a) and Jurkat (b) cells were cotransfected with PGL3-2.6, PGL3-1.5, or PGL3-320, the internal control pRLTK, and either the Tax expression plasmid pCA-Tax 1-HIS or empty pCA vector. Luciferase activity was quantified after 48 h and was normalized to TK activity. Basal activity was constant for all of the reporters and was set at 100%. Differences in normalized luciferase activity were significant as determined by Student's t test. (c) 293T cells were cotransfected with the indicated concentrations of pCA-Tax 1-HIS or empty pCA vector together with the PDGF luciferase reporter pRALuc (35) and the internal control pRLTK. Luciferase activity was quantified after 24 h and was normalized to TK activity. Basal activity was set at 100%.

FIG. 4.

Sp1 relieves Tax 1-induced downregulation of the VEGF promoter. (a) 293T cells were cotransfected with PGL3-320 (Sp1 sites only) and increasing amounts of Sp1 expression vector or empty pCA vector. Transfections were carried out in triplicate. Luciferase activity was quantified 48 h after transfection and basal activity in the absence of exogenous Sp1 was set at 100%. The expression levels of Sp1 was detected in lysates by Western blotting using anti-Sp1 antibody (Calbiochem) and is shown in the lower panel. (b) 293T cells were cotransfected with PGL3-320, 600 ng pCA-Tax 1-HIS and increasing amounts of pCA-Sp1, or empty pCA plasmid. Transfections were carried out in triplicate. Forty-eight hours after transfection, luciferase activity was quantified and basal activity is set at 100% (last bar). The expression levels of Tax 1 and Sp1 in lysates from cells transfected with 600 ng of Tax 1 and 200 ng (lane 1), 300 ng (lane 2), 400 ng (lane 3), 500 ng (lane 4), 600 ng (lane 5) of Sp1, or the relevant empty plasmids (lane 6) was detected by Western blotting using anti-Tax 1 and anti-Sp1 antibody (Calbiochem), as shown in the lower panel.

FIG. 5.

Tax 1 reduces Sp1 binding to the VEGF promoter. Electromobility shift assays were performed on nuclear extract (NE) obtained from pCA-Tax1-HIS-transfected and nontransfected 293T cells (a) and CEM and MT2 cells (b) using a biotin-labeled Sp1 consensus probe. Bound complexes were detected using chemiluminescence. Unlab, unlabeled. (c) The expression levels of Tax 1 in the nuclear extracts used in EMSAs were detected by Western blotting using anti-Tax 1. The levels of PARP (poly (ADP-ribose) polymerase 1) are shown as a loading control (lower panel).

FIG. 6.

Levels of VEGF secreted by Tax-positive and -negative cell lines. (a) 293T cells were transfected with 1 μg of pCA-Tax1-HIS or pCA empty. Cells were incubated for 48 h, and VEGF was quantified in cell culture supernatants by ELISA. (b) A total of 1 × 10⁶ cells each of the MT2 and C91 (HTLV-1 transformed), H2 (HTLV-2 transformed), TH, TE, CR (ATL cell lines), and CEM and Jurkat cells lines was cultured for 48 h, and VEGF protein in cell culture supernatant was quantified by ELISA.