Elsevier

Leukemia Research

Volume 34, Issue 6, June 2010, Pages 763-768
Leukemia Research

Transactivation of human osteopontin promoter by human T-cell leukemia virus type 1-encoded Tax protein

https://doi.org/10.1016/j.leukres.2009.08.028Get rights and content

Abstract

Osteopontin (OPN) is a cytokine that contributes substantially to the growth and metastasis in a wide spectrum of malignancies. We report here that OPN gene is transactivated by Tax protein of human T-cell leukemia virus type 1 (HTLV-1). Northern blot showed enhanced OPN gene expression in cells stably expressing Tax. Co-expression of Tax increased the reporter gene expression directed by OPN promoter. Tax-induced OPN activation was abrogated by treatment with LY294002 (PI3K inhibitor) or co-transfection with AKT siRNA, suggesting PI3K/AKT pathway is involved in Tax-mediated transactivation. Reporter assay with deletion mutants showed that the 5′-partial sequence between −765 and −660 of the OPN promoter is the region responsive to Tax, and further, disrupting the AP-1 site within this region abolished the OPN induction by Tax, indicating that Tax activation of OPN promoter is likely mediated by AP-1 site. This study suggests that OPN is one of the downstream mediators of aberrantly activated PI3K/AKT signaling by Tax, which may partially contribute to HTLV-1-associated leukemogenesis.

Introduction

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus etiologically linked to the development of adult T-cell leukemia (ATL) [1] and to chronic inflammatory disease such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM-TSP) [2], [3]. Although the mechanisms of HTLV-1-associated leukemogenesis are not yet fully understood, there is strong accumulating evidence demonstrating that viral oncoprotein Tax plays an essential role in the oncogenic process [4], [5], [6]. Tax protein is crucial for initiating cellular transformation, although it may be dispensable for maintenance of transformation. Tax initiated transformation has been shown to start with mitogenic activation of lymphocytes in G1-phase with concomitant perturbed tumor suppressor function and enhanced survival function [7]. Tax contributes to cellular transformation through alteration of cellular gene expression and dysregulation of cellular signal pathways important for cell cycle progression and cell survival [8]. In addition, Tax also facilitates cell proliferation and transformation by interacting with and modulating activity of numerous cellular proteins. For instance, Tax interacts with cell cycle associated protein such as cyclin D, cyclin-dependent kinase (CDK) and CDK inhibitors, leading to an increased G1-S phase transition efficiency. Likewise, Tax was shown to bind and inactivate tumor suppressors such as Dlg and consequently accelerates uncontrolled cell growth. Moreover, Tax targets multiple components of DNA damage repair pathway and silences cellular checkpoints, thereby attenuating G2/M arrest and apoptosis [see details in the review of Ref. [9]]. Several studies have demonstrated that AKT is activated in HTLV-1 transformed cells and that Tax activation of AKT is linked to apoptosis inhibition and cell survival [10], [11]. AKT, also known as protein kinase B, is a major downstream target of PI3K-triggered signaling. AKT is activated by PI3K via site-specific phosphorylation. The PI3K/AKT pathway has been shown to be a key regulator of a variety of cellular events including cell survival and proliferation [12], [13], [14]. The proliferative effect of AKT occurs by triggering multiple transcriptional factors, such as AP-1. AP-1 is an important transcription factor that regulates the expression of many genes. The major form of AP-1 in most cells is heterodimer composed of fos and jun, which have high affinity to bind to AP-1 element. It was previously reported that Tax, independent of its ability to stimulate NF-κB signaling, activates AP-1 via PI3K/AKT pathway, which consequently provokes uncontrolled cell proliferation and malignant transformation [15].

Osteopontin (OPN) is a secreted phosphoprotein produced by osteoclasts, macrophages, T-cells, kidneys, and vascular smooth muscle cells. Physiologically, OPN is an essential acute-phase cytokine that mediates the homing of immune system cells and elicits protective immunity against foreign pathogens [16]. In addition to its role in immune response, another interesting facet of OPN is its participation in tumor progression and metastasis. By interacting with its receptors, CD44 and αvβ3 integrin, OPN promotes migration and invasion of tumor cells [17], [18], inhibits apoptosis [19], facilitates extracellular matrix remolding [20] and angiogenesis [21], [22]. Consistent with the experimental observations, clinical statistical data also showed a correlation between OPN level and the metastatic outcome in breast cancer [23]. OPN is responsive to various cell growth signals, including estrogen and progesterone, calcitriol, and 12-O-tetradecanoylphorbol-13-acetate [24], [25], [26]. Transcriptional regulation of OPN is complex and involves multiple transcription factors, including AP-1, Ets, Myc, and v-Src [27], [28], [29].

We previously observed an elevated plasma level of OPN in HTLV-1-associated ATL, especially in acute-type ATL (data not shown). In this study, we traced the cause for this observation and found that enhanced OPN expression is attributable, at least partially, to transactivating effect of Tax on OPN promoter. The augmentation of OPN transcription by Tax appears to be mediated by PI3K/AKT pathway and a distal AP-1 element in OPN promoter. These findings contribute incrementally to further our understanding of how Tax oncoprotein plays a crucial role in HTLV-1-related oncogenesis.

Section snippets

Plasmids

Control plasmid pCn, and plasmids encoding wild-type HTLV-1 Tax (pCnwtax) or its mutants (pCnm148 and pCnm319) have all been described previously [30], [31]. For construction of pOPN1-luc, the promoter sequence from −765 to +23 was amplified by genomic PCR using human genomic DNA as a template with the primers 5′-ctcggtaccTAGGTAATAGTATGCA-3′ and 5′-atagagctcTGCCTCCTCCTGCT-3′. Various size deletions of the promoter were also generated by PCR with various 5′ primers and the same fixed 3′ primer.

Tax induces up-regulated transcription of OPN

Our unpublished data showed an elevated plasma level of OPN in HTLV-1-associated ATL, especially in acute-type ATL (data not shown). This result promoted us to examine the possible role of Tax in up-regulated expression of OPN in ATL. For this purpose, total RNA was prepared from Huhwtax stably expressing Tax, which was established by transfection with pCnwtax followed by G418 selection, and subjected to Northern blot analysis with full-length labeled OPN as a probe. HuhCn cells stably

Discussion

In this study, we set out to investigate the regulation of OPN, a gene important for tumor progression and metastasis, by HTLV-1 Tax. The increase of endogenous OPN RNA level in the presence of Tax correlated well with the enhanced OPN promoter activity in reporter assay, indicating that Tax acts by stimulating the initiation of OPN mRNA synthesis. Unlike the wild-type Tax, neither of the mutant m148 (NF-κB/CREB+) and m319 (NF-κB+/CREB) was able to fully activate the promoter, suggesting that

Conflict of interest

The authors declare that they have no potential conflicts of interest.

Acknowledgements

We thank Dr. Masahiro Fujii for Tax2A- and Tax2B-expressing plasmids, and Dr. Shoji Yamaoka for wild- and mutant-type Tax-expressing constructs. This work was supported in part by Grant-in-Aid for Scientific Research from JSPS and the Scientific Research Expenses for Health and Welfare Program from the Ministry of Health and Welfare, Japan.

Contributions. All authors contributed to either the design or implementation of the studies described in this manuscript.

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