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Human T-cell Leukemia Virus Type I Tax Down-regulates the Expression of Phosphatidylinositol 3,4,5-Trisphosphate Inositol Phosphatases via the NF-κB Pathway

2008; Elsevier BV; Volume: 284; Issue: 5 Linguagem: Inglês

10.1074/jbc.m806325200

1083-351X

Ryu-ich Fukuda, Kiyohito Tsuchiya, Koji Suzuki, Katsuhiko Itoh, Jun Fujita, Atae Utsunomiya, Takashi Tsuji,

Animal Disease Management and Epidemiology

Human T-cell leukemia virus type I (HTLV-I) is an oncogenic retrovirus that causes adult T-cell leukemia/lymphoma (ATLL). The virus encodes an oncoprotein, Tax, which functions in transcriptional regulation, cell cycle control, and transformation. Through its pleiotropic actions, Tax plays critical roles in leukemogenesis. We have previously reported that PTEN and SHIP-1, PIP3 inositol phosphatases that negatively regulate the phosphatidylinositol (PI) 3-kinase signaling cascade, are disrupted in ATLL neoplasias. Overactivation of PI3-kinase signaling has an essential role in both development of ATLL-specific nuclear polymorphisms and onset of ATLL. We report here that both PTEN and SHIP-1 are down-regulated by HTLV-I Tax through the NF-κB signaling pathway. Tax expression up-regulated phosphorylated Akt, a downstream serine/threonine kinase in the PI3-kinase signaling cascade. Transduction of NF-κB p65, which mimics the activation of NF-κB signaling, also suppressed these phosphatases. An IκBΔN mutant that inhibits the activation of NF-κB prevented PIP3 phosphatase down-regulation by Tax. The underlying mechanism of NF-κB-mediated suppression of PIP3 phosphatases involved sequestration of the coactivator p300 by p65. These down-regulations of PIP3 phosphatases were found to be essential for the Tax-induced cell proliferation. Thus, our results suggest that HTLV-I Tax down-regulates PIP3 phosphatases through the NF-κB pathway, resulting in increased activation of the PI3-kinase signaling cascade in human T-cells and contributing to leukemogenesis. Human T-cell leukemia virus type I (HTLV-I) is an oncogenic retrovirus that causes adult T-cell leukemia/lymphoma (ATLL). The virus encodes an oncoprotein, Tax, which functions in transcriptional regulation, cell cycle control, and transformation. Through its pleiotropic actions, Tax plays critical roles in leukemogenesis. We have previously reported that PTEN and SHIP-1, PIP3 inositol phosphatases that negatively regulate the phosphatidylinositol (PI) 3-kinase signaling cascade, are disrupted in ATLL neoplasias. Overactivation of PI3-kinase signaling has an essential role in both development of ATLL-specific nuclear polymorphisms and onset of ATLL. We report here that both PTEN and SHIP-1 are down-regulated by HTLV-I Tax through the NF-κB signaling pathway. Tax expression up-regulated phosphorylated Akt, a downstream serine/threonine kinase in the PI3-kinase signaling cascade. Transduction of NF-κB p65, which mimics the activation of NF-κB signaling, also suppressed these phosphatases. An IκBΔN mutant that inhibits the activation of NF-κB prevented PIP3 phosphatase down-regulation by Tax. The underlying mechanism of NF-κB-mediated suppression of PIP3 phosphatases involved sequestration of the coactivator p300 by p65. These down-regulations of PIP3 phosphatases were found to be essential for the Tax-induced cell proliferation. Thus, our results suggest that HTLV-I Tax down-regulates PIP3 phosphatases through the NF-κB pathway, resulting in increased activation of the PI3-kinase signaling cascade in human T-cells and contributing to leukemogenesis. Human T-cell leukemia virus type I Tax down-regulates the expression of phosphatidylinositol 3,4,5-trisphosphate inositol phosphatases via the NF-κB pathway.Journal of Biological ChemistryVol. 286Issue 49PreviewVOLUME 284 (2009) PAGES 2680–2689 Full-Text PDF Open Access Human T-cell leukemia virus type I (HTLV-I) 2The abbreviations used are: HTLV, human T-cell leukemia virus type; PIP3, phosphatidylinositol 3,4,5-trisphosphate; ATLL, adult T-cell leukemia/lymphoma; PI, phosphatidylinositol; CREB, cyclic AMP-responsive element-binding protein; GFP, green fluorescent protein; mAb, monoclonal antibody; pAb, polyclonal antibody; KLH, keyhole limpet hemocyanin; HA, hemagglutinin; LTR, long terminal repeat; PBT, peripheral blood T-cell; p300wt, p300 wild type; IL, interleukin; CBP, CREB-binding protein. preferentially infects and subsequently transforms T-cells into acute malignant cells, resulting in adult T-cell leukemia/lymphoma (ATLL) (1Uchiyama T. Yodoi J. Sagawa K. Takatsuki K. Uchino H. Blood.. 1977; 50: 481-492Google Scholar, 2Yoshida M. Miyoshi I. Hinuma Y. Proc. Natl. Acad. Sci. U. S. A... 1982; 79: 2031-2035Google Scholar). ATLL generally presents following prolonged incubation periods ranging from 40 to 60 years. The vast majority of infected individuals remain clinically asymptomatic, with only 2–5% developing neoplasia. An accumulation of leukemogenic events within HTLV-I-infected T-cells are required for the development of ATLL (2Yoshida M. Miyoshi I. Hinuma Y. Proc. Natl. Acad. Sci. U. S. A... 1982; 79: 2031-2035Google Scholar, 3Yoshida M. Annu. Rev. Immunol... 2001; 19: 475-496Google Scholar, 4Shimoyama M. Minato K. Tobinai K. Nagai M. Setoya T. Takenaka T. Ishihara K. Watanabe S. Hoshino H. Miwa M. Kinoshita M. Okabe S. Fukushima N. Inada N. Jpn. J. Clin. Oncol... 1983; 13: 165-187Google Scholar, 5Yasunaga J. Matsuoka M. Int. J. Hematol... 2003; 78: 312-320Google Scholar). However, the molecular basis for this accumulation remains unclear. The diverse clinical features of this disease led to its subclassification into acute, lymphoma, chronic, and smoldering subtypes. In acute type ATLL patients, peripheral T-cells display a typical multilobulated nuclear appearance (flower-like nuclei) (4Shimoyama M. Minato K. Tobinai K. Nagai M. Setoya T. Takenaka T. Ishihara K. Watanabe S. Hoshino H. Miwa M. Kinoshita M. Okabe S. Fukushima N. Inada N. Jpn. J. Clin. Oncol... 1983; 13: 165-187Google Scholar, 6Shimoyama M. Br. J. Haematol... 1991; 79: 428-437Google Scholar). In addition to the structural gag and env genes and retroviral enzymes, the HTLV-I genome has a region at its 3′ end that was originally designated as the pX region. The proteins encoded in pX region regulate not only viral gene expression but also the expression of a large variety of cellular genes. This pX region encodes some nonstructural proteins, including Tax, Rex, and certain accessory proteins (7Nicot C. Harrod R.L. Ciminale V. Franchini G. Oncogene.. 2005; 24: 6026-6034Google Scholar). Tax is recognized as the most notable viral oncoprotein. Recent studies demonstrated that Tax plays central roles in tumorigenesis and contributes to its own pathogenesis through its capacity to immortalize primary T-cells, to transform rodent fibroblasts, and to induce tumors in transgenic mice expressing Tax (8Rosin O. Koch C. Schmitt I. Semmes O.J. Jeang K.T. Grassmann R. J. Biol. Chem... 1998; 273: 6698-6703Google Scholar, 9Liu Y. Wang Y. Yamakuchi M. Masuda S. Tokioka T. Yamaoka S. Maruyama I. Kitajima I. Oncogene.. 2001; 20: 2514-2526Google Scholar, 10Hasegawa H. Sawa H. Lewis M.J. Orba Y. Sheehy N. Yamamoto Y. Ichinohe T. Tsunetsugu-Yokota Y. Katano H. Takahashi H. Matsuda J. Sata T. Kurata T. Nagashima K. Hall W.W. Nat. Med... 2006; 12: 466-472Google Scholar). The majority of the oncogenic properties of Tax are related to its ability to activate the expression of cellular genes that control T-cell proliferation and differentiation through induction of the constitutive activation of NF-κB, a transcriptional regulator of numerous cellular genes associated with diverse central biological processes (11Sun S.C. Yamaoka S. Oncogene.. 2005; 24: 5952-5964Google Scholar). Activation of the IκB kinase complex by Tax regulates the phosphorylation of the IκB proteins that inhibit NF-κB, resulting in their ubiquitination and degradation by the proteasome and the subsequent transition of NF-κB complexes into the nucleus (11Sun S.C. Yamaoka S. Oncogene.. 2005; 24: 5952-5964Google Scholar, 12Siebenlist U. Brown K. Claudio E. Nat. Rev. Immunol... 2005; 5: 435-445Google Scholar). Tax also indirectly activates transcription by recruiting or modifying the activity of cellular transcription factors, including cyclic AMP-responsive element-binding protein (CREB), serum-responsive factor, and NF-κB. Tax is also reported to directly activate oncogenes and inactivate tumor suppressor genes (3Yoshida M. Annu. Rev. Immunol... 2001; 19: 475-496Google Scholar, 13Grassmann R. Aboud M. Jeang K.T. Oncogene.. 2005; 24: 5976-5985Google Scholar). In T-cells, NF-κB indirectly promotes T-cell activation and proliferation through the induction of immune regulatory cytokines. In addition, NF-κB transcriptional regulation directly modulates T-cell function by regulating cell survival through the induction of Bcl-xL and cIAP and the cell cycle through the induction of cyclinD1, cyclinD2, and c-Myc. It is thought that over-activation of NF-κB leads to abnormal regulation of gene expression, and NF-κB is involved in oncogenesis. NF-κB is known to bind to the multifunctional protein CBP/p300. CBP/p300 functions as a scaffold protein in multicomponent transcriptional regulatory complexes, contributing to the activity of DNA-binding transcription factors, and as a histone acetyltransferase, influencing chromatin activity by modulating nucleosomal histones (14Chan H.M. La Thangue N.B. J. Cell Sci... 2001; 114: 2363-2373Google Scholar). Several lines of evidence indicate that CBP/p300 proteins are rate-limiting for transcription. Competition for a limited amount of CBP/p300 upon NF-κB activation has been previously proposed as a mechanism for either transcriptional regulation or induction of apoptosis (15Horvai A.E. Xu L. Korzus E. Brard G. Kalafus D. Mullen T.M. Rose D.W. Rosenfeld M.G. Glass C.K. Proc. Natl. Acad. Sci. U. S. A... 1997; 94: 1074-1079Google Scholar, 16Hottiger M.O. Nabel G.J. J. Virol... 1998; 72: 8252-8256Google Scholar, 17Kamei Y. Xu L. Heinzel T. Torchia J. Kurokawa R. Gloss B. Lin S.C. Heyman R.A. Rose D.W. Glass C.K. Rosenfeld M.G. Cell.. 1996; 85: 403-414Google Scholar, 18Nicot C. Harrod R. Mol. Cell. Biol... 2000; 20: 8580-8589Google Scholar, 19Webster G.A. Perkins N.D. Mol. Cell. Biol... 1999; 19: 3485-3495Google Scholar). The sequestration of CBP/p300 by NF-κB also down-regulates the transcriptional activity of several transcription factors, including p53 and c-Myb (20Wadgaonkar R. Phelps K.M. Haque Z. Williams A.J. Silverman E.S. Collins T. J. Biol. Chem... 1999; 274: 1879-1882Google Scholar, 21Nicot C. Mahieux R. Pise-Masison C. Brady J. Gessain A. Yamaoka S. Franchini G. Mol. Cell. Biol... 2001; 21: 7391-7402Google Scholar). Abnormal regulation of CBP/p300 by NF-κB is thought to have an important role in the oncogenesis. The PI3-kinase signaling cascade is essential for a variety of biological processes, including proliferation, T-cell activation, regulation of apoptosis, and cell movement (22Deane J.A. Fruman D.A. Annu. Rev. Immunol... 2004; 22: 563-598Google Scholar). PI3-kinase activity produces phosphatidylinositol 3,4,5-trisphosphate (PIP3) by the phosphorylation of phosphatidylinositol 4,5-bisphosphate, which subsequently regulates downstream effector molecules. In contrast, PIP3 phosphatases, such as the PTEN (phosphatase and tensin homolog deleted on chromosome 10) tumor suppressor and SHIP-1 (Src homology 2 domain containing inositol polyphosphate phosphatase-1), which dephosphorylate PIP3 and act as negative regulators, modulate various biological phenomena (22Deane J.A. Fruman D.A. Annu. Rev. Immunol... 2004; 22: 563-598Google Scholar). The alteration of PI3-kinase signaling components through either activation of oncogenes or inactivation of tumor suppressors disrupts a signaling equilibrium and can thus lead to cellular transformation (23Dillon R.L. White D.E. Muller W.J. Oncogene.. 2007; 26: 1338-1345Google Scholar). Recently, we demonstrated that alteration of the signaling cascade through PIP3 phosphatase disruption was essential for ATLL-type multilobulated nuclear formation, an important diagnostic marker and indicator for development of ATLL via abnormal T-cell proliferation (24Fukuda R. Hayashi A. Utsunomiya A. Nukada Y. Fukui R. Itoh K. Tezuka K. Ohashi K. Mizuno K. Sakamoto M. Hamanoue M. Tsuji T. Proc. Natl. Acad. Sci. U. S. A... 2005; 102: 15213-15218Google Scholar). Recent studies demonstrated that Tax induces abnormal cell growth through the production of PIP3 and/or the activation of a downstream effector of PI3-kinase, the kinase Akt (25Peloponese Jr., J.M. Jeang K.T. J. Biol. Chem... 2006; 281: 8927-8938Google Scholar, 26Okamoto N. Tezuka K. Kato M. Abe R. Tsuji T. Biochem. Biophys. Res. Commun... 2003; 310: 691-702Google Scholar). However, how Tax modulates PIP3 production and the mechanism of Akt signaling activation has not been established. Current evidence suggests the possibility that abnormal regulation of the PI3-kinase signaling cascade in ATLL-T cells and Tax-expressing cells may be common underlying mechanisms in altered PIP3 phosphatase gene expression. In this study, we show that Tax down-regulates the expression of PIP3 phosphatases through NF-κB activation, leading to the overactivation of PI3-kinase signaling cascade in T-cells. We also report that activated NF-κB directly binds to p300 and influences the expression of not only PTEN but also SHIP-1 as a result of p300 sequestration by activated NF-κB. These results suggest that the mechanism of Tax-regulated alteration of the PI3-kinase signaling cascade involves suppression of PIP3 phosphatase gene expression through NF-κB-mediated inhibition of p300 recruitment. We also propose putative molecular mechanisms for Tax-mediated alteration of the PI3-kinase signaling cascade. Cell Culture and Treatment—Highly purified T-cells (>97%) were isolated from healthy adult volunteers following informed consent as described previously (26Okamoto N. Tezuka K. Kato M. Abe R. Tsuji T. Biochem. Biophys. Res. Commun... 2003; 310: 691-702Google Scholar). This study was approved by the Ethics Committee of Tokyo University of Science. JPX-9 cells, which were kindly provided by Dr. K. Sugamura (Tohoku University), are derivatives of the Jurkat human T-cell line that have a stably integrated Tax gene under the control of a metallothionein promoter JPX-9 cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin (27Nagata K. Ohtani K. Nakamura M. Sugamura K. J. Virol... 1989; 63: 3220-3226Google Scholar). For the induction of Tax, the cells were cultured with 10 μm of CdCl2. Retroviral Gene Transfer—pMX retroviral vectors and Plat-A packaging cells were kindly supplied by Dr. T. Kitamura (University of Tokyo) (28Onishi M. Kinoshita S. Morikawa Y. Shibuya A. Phillips J. Lanier L.L. Gorman D.M. Nolan G.P. Miyajima A. Kitamura T. Exp Hematol... 1996; 24: 324-329Google Scholar). Plat-A cells were maintained in high glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. The cells were passaged at equivalent cell numbers into a 100-mm culture dish 48 h before transfection. After reaching ∼50% confluence, the cells were transfected with plasmids that had been preincubated for 30 min with 100 μl of serum-free medium containing FuGENE 6 (Roche Applied Science). Immunoblotting Analyses—Immunoblotting was performed according to methods described previously (26Okamoto N. Tezuka K. Kato M. Abe R. Tsuji T. Biochem. Biophys. Res. Commun... 2003; 310: 691-702Google Scholar). The cells were washed in phosphate-buffered saline and lysed in lysis buffer (50 mm Tris-HCl, pH 7.5, 1 mm EGTA, 5 mm EDTA, 1% Triton X-100, 50 mm NaF, 0.2 mm Na3VO4) in the presence of protease inhibitors (0.1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 μg/ml aprotinin). The cell extracts were incubated on ice for 15 min and centrifuged (10,000 × g) at 4 °C for 10 min, and then the supernatants were collected. Tris-Glycine gels, 4–20% (Daiichi Chemicals, Tokyo, Japan), were used as recommended by the manufacturer. The proteins were then transferred to polyvinylidene difluoride membranes (Daiichi Chemicals), membrane nonspecific binding sites were blocked for 30 min in nonfat dry-milk or bovine serum albumin at room temperature, and membrane-bound proteins were analyzed using specific antibodies. Antibodies included anti-GFP polyclonal antibody (pAb), are purified from rabbit serum using ion exchange chromatography (MBA, Nagoya, Japan), anti-PTEN pAb, which are produced by immunizing rabbit with a synthetic peptide (KLH-coupled) derived from the sequence of human PTEN (Cell Signaling Technology, Danvers, MA), anti-SHIP-1 pAb (D1163; Cell Signaling Technology), anti-phospho-Akt/PKB pAb, which are produced by immunizing rabbits with a synthetic phospho-peptide (KLH-coupled) corresponding to residues surrounding Ser473 of mouse Akt (Cell Signaling Technology), anti-Akt/PKB pAb, which are produced by immunizing rabbit with a synthetic peptide (KLH-coupled) derived from the carboxyl-terminal sequence of mouse Akt (Cell Signaling Technology), anti-p65 pAb, which are produced by immunizing rabbit with a synthetic peptide (KLH-coupled) derived from the carboxyl-terminal sequence of human p65 (Upstate Biotechnology, Inc., Lake Placid, NY), anti-p300 mAb (NM11; BD Bioscience, San Jose, CA), anti-HA pAb, which are produced by immunizing rabbit with a synthetic peptide corresponding to amino acid residues 98–106 (YPYDVPDYA) of human influenza virus hemagglutinin (HA) known as HA tag, conjugated to KLH (Sigma) or anti-FLAG mAb (M2; Sigma) according to the manufacturer's instructions. After washing membranes, bound primary antibodies were visualized with a horseradish peroxidase-conjugated F(ab′)2 fragment against rabbit IgG (ICN/Cappel, Aurora, OH) using ECL (Amersham Biosciences). Immunoprecipitation—For the immunoprecipitation of p65 and p300, the cell lysates were incubated with anti-p65 pAb (Upstate Biotechnology) and anti-p300 (BD Bioscience) and incubated with protein-G-Sepharose beads (Amersham Biosciences, England). The beads were washed with lysis buffer, and the bound proteins were solubilized by boiling for 5 min in SDS sample buffer. Immunoblotting was performed according to methods described. The membranes were then incubated with anti-GFP pAb (MBL), anti-HA pAb (Sigma), anti-FLAG pAb (Sigma), anti-p65 pAb (Upstate Biotechnology), and anti-p300 mAb (BD Bioscience). After washing, the bound primary antibodies were detected as described above. Transient Transfection and Reporter Assay—Transfections were performed using early passage JPX-9 cells and activated PBT. Transfections were carried out using the Nucleofector Kit V or Kit T (Amaxa, Koeln, Germany) and a Nucleofector apparatus (Amaxa) according to the manufacturer's instructions. Tax-GFP, GFP-p65, GFP-p300, His-p300, GFP-p300N, FLAG-p300N, HA-p300ΔN, and GFP-p300ΔN cDNA were used for transfection. 48 h after transfection, the GFP-positive cells were sorted by EPICS ALTRA (Beckman Coulter, Fullerton, CA). Luciferase reporter vectors containing NF-κB binding sites (pNF-κB-Luc) in the promoter (Stratagene, La Jolla, CA) were cotransfected with Tax-GFP. 36 h after transfection, the cells were harvested, washed in phosphate-buffered saline, solubilized in 100 μl of reporter lysis buffer (Dual Luciferase Reporter Assay; Promega Biotech, Madison, WI), and assayed for luciferase activity using a ARVO Light (PerkinElmer Life Sciences). RNA Isolation and Real Time Reverse Transcription-PCR—The expression of PTEN, SHIP-1, Bcl-xL, and β-actin were assessed by real time reverse transcription PCR. Total RNA was prepared from cells under various conditions using TRIzol reagent (Invitrogen). First strand cDNAs were synthesized using random primers and a first strand cDNA synthesis kit (Toyobo, Tokyo, Japan). PCR mixtures were prepared using SYBR Premix Ex Taq (Takara, Tokyo, Japan) containing 0.2 mm of each primer, and amplification reactions were performed. The sequences of PTEN, SHIP-1, and Bcl-xL primer pairs were: PTEN (forward, 5′-ccaatgttcagtggcggaact-3′; reverse, 5′-gaacttgtcttcccgtcgtgtg-3′); SHIP-1 (forward, 5′-cgacaagaagcctgagtcccttt-3′; reverse, 5′-ggtagttaagatccccaaaccagaa-3′); Bcl-xL (forward, 5′-gcggctgggatacttttctg-3′; reverse 5′-cgttgctggccctttcg-3); and β-actin (forward, 5′-gccacggtcgcttcca-3′; reverse, 5′-gaaccgctcattgccaatg-3′). The gene expression levels of PTEN, SHIP-1, and Bcl-xL were measured using the ABI Prism 7000 sequence detection system (Applied Biosystems). PCR product levels were estimated by the measurement of the intensity of fluorescence of SYBR Green. Gene expression levels were normalized to β-actin mRNA expression. Plasmid Construction—All of the constructs were verified by DNA sequencing. The Tax insert was created by reverse transcription-PCR from JPX-9 cell cDNA. The Tax plasmid was produced by ligation of the full-length Tax cDNA sequence (Eco47III/NotI fragment) into pEGFP-C1 (BD Clontech, Palo Alto, CA). The primers used for amplification for Tax cDNA were: forward, 5′-aaagaattccatggcccacttcccagggt-3′; reverse, 5′-aaagtctacgcgacttctgtttcgcggaa-3′. The Tax M22 plasmid, with amino acid substitutions at residues 130 and 131 converting Thr-Leu to Ser-Ala, was produced from the Tax plasmid using the QuikChange II site-directed mutagenesis kit (Stratagene). The Tax-GFP fusion and Tax M22-GFP fusion cDNA sequences (Eco47III/NotI fragment) were cloned into the pMX vector. The IκBΔN plasmid was subcloned into pMX using EcoRI and NotI restriction sites. The p65 plasmid was created by reverse transcription-PCR from human peripheral blood T-cell cDNA and cloned into the pEGFP-C1 (BD Clontech) vector using XhoI and BamHI restriction sites, and the p65-GFP fusion sequence (Eco47III/BamHI fragment) was cloned into the pMxs vector. The primers used for amplification for p65 cDNA were: forward, 5′-aaactcgagccaccatggacgaactgttccc-3′; reverse, 5′-aaaggatccgctgcggagctgatctgactcagcag-3′. The p300 plasmid was subcloned into the pMX vector using XhoI and SalI restriction sites. The HTLV-1 LTR reporter plasmid was kindly provided by K. Shimotohno (Kyoto University) (29Youn H.G. Matsumoto J. Tanaka Y. Shimotohno K. J. Virol... 2003; 77: 10015-10027Google Scholar). p300N plasmid was subcloned into pIRES-hrGFP1a vector using NotI/BamHI site. p300ΔN plasmid was subcloned into pMx vector using XhoI/SalI site, and HA tag fragment was inserted using NheI/SalI site. Fluorescence Microscopy—The cells were stained with 10 μg/ml of anti-p65 pAb (Upstate Biotechnology) and then stained fluorescein isothiocyanate-conjugated anti-rabbit IgG goat (Fab′)2 fragments and Hoechst dye 33258 (Sigma). After staining, the cells were analyzed by fluorescent microscopy using an Axiovert 200M (Carl Zeiss, Jena, Germany). Image acquisition was made with an AxioCAM MRm (Carl Zeiss), and the images were processed with AxioVision software (Carl Zeiss). Short Hairpin RNA Constructs—p300-specific knockdowns in JPX-9 cells were performed by the expression of hairpin siRNA using a pSuper.neo+gfp in pSuper RNA interference system (OligoEngine, Seattle, WA). To generate the p300- and control luciferase-RNA interference plasmid, a unique nucleotide sequence p300 (5′-gcggctgggatacttttctg-3′) and luciferase (5′-cgtacgcgggaatacttcga-3′), derived from the mRNA transcript of p300 and luciferase, were synthesized and cloned into pSuper.neo+gfp vector according to the manufacturer's instructions. Cell Proliferation Assay—Cell proliferation was determined by using either direct cell counting or the Cell Counting Kit-8 (Dojindo Labs, Tokyo, Japan) according to the manufacturer's protocol. After transfection, the cells were plated in a 96-well plate and cultured for 48 h. WST-8 reagent (Dojindo Labs) was added to each well and incubated for 1–4 h. The cell viability in each well was determined by reading the optical density at 450 nm. HTLV-1 Tax Induces the Activation of PI3-Kinase/Akt Pathway via the Down-regulation of PTEN and SHIP-1—It has been reported that alteration of PI3-kinase signaling cascade induced abnormal cell proliferation in both ATLL cells and Tax-expressing cells (9Liu Y. Wang Y. Yamakuchi M. Masuda S. Tokioka T. Yamaoka S. Maruyama I. Kitajima I. Oncogene.. 2001; 20: 2514-2526Google Scholar, 24Fukuda R. Hayashi A. Utsunomiya A. Nukada Y. Fukui R. Itoh K. Tezuka K. Ohashi K. Mizuno K. Sakamoto M. Hamanoue M. Tsuji T. Proc. Natl. Acad. Sci. U. S. A... 2005; 102: 15213-15218Google Scholar, 25Peloponese Jr., J.M. Jeang K.T. J. Biol. Chem... 2006; 281: 8927-8938Google Scholar). To study the mechanism of this regulation, we analyzed the role of Tax in regulating the expression of PTEN and SHIP-1 in peripheral blood T-cells (PBT) isolated from healthy donors that has been activated by CD3 and CD28 stimulation. We found that transduction of Tax significantly reduced the expression of not only PTEN but also SHIP-1 proteins by Western blot analysis (Fig. 1A). The expression of Tax protein also significantly decreased the expression of these PIP3-phosphatase mRNAs in activated PBT (Fig. 1B). JPX-9 cells, which were transfected with a Tax gene regulated by a metallothionein promoter and inducible by CdCl2, were commonly used for Tax-function analysis (27Nagata K. Ohtani K. Nakamura M. Sugamura K. J. Virol... 1989; 63: 3220-3226Google Scholar). When JPX-9 cells were treated with CdCl2, the expressions of the PIP3-phosphatase mRNAs were significantly decreased (data not shown). We also transfected with Tax cDNA to JPX-9 cells and analyzed the effect of the expressions of those mRNAs whether the reduction of those mRNA expressions induced by CaCl2 could be due to the direct effect by the Tax expression. Not only PTEN mRNA but also SHIP-1 mRNA were also significantly decreased the transduction of Tax in JPX-9 cells (Fig. 1B). Next, we determined the effects of Tax expression on AKT, a downstream effector of PI3-kinase, in activated PBT. We found that Tax induced the phosphorylation of Akt. Interestingly, phosphorylated Akt (p-Akt) levels were inversely correlated with PTEN and SHIP-1 expression (Fig. 1C). These results indicate that Tax expression induced the activation of the PI3-kinase/Akt signaling pathway through the down-regulation of PIP3 phosphatases PTEN and SHIP-1. NF-κB Activation Motif in Tax Is Essential for the Down-regulation of PIP3 Phosphatases by Tax—It is well known that Tax modulates gene expression and associated cellular events through the constitutive activation of NF-κB (11Sun S.C. Yamaoka S. Oncogene.. 2005; 24: 5952-5964Google Scholar). We next examined the involvement of NF-κB activation in Tax-mediated down-regulation of PIP3 phosphatase mRNAs. In our experiments using the activated PBT and JPX-9 cells, Tax expression induced the nuclear localization of p65 and the transactivation subunit of NF-κB complexes and up-regulated the expression of a downstream target gene, Bcl-xL, which is served as a positive control (Fig. 2A) (30Nakano H. Nakajima A. Sakon-Komazawa S. Piao J.H. Xue X. Okumura K. Cell Death Differ.. 2006; 13: 730-737Google Scholar, 31Karin M. Greten F.R. Nat. Rev. Immunol... 2005; 5: 749-759Google Scholar). We also examined the activation of NF-κB using NF-κB-Luc, a reporter assay that utilizes luciferase regulated by a NF-κB-binding promoter. We found that Tax expression induced in JPX-9 cells by CdCl2 treatment resulted in NF-κB DNA binding activity (Fig. 2A, right panels). We next investigated whether a Tax M22 mutant (T130L131-A130S131), which lacks the ability to activate the NF-κB pathway but retains the ability to transactivate the CREB-dependent HTLV-I LTR (32Smith M.R. Greene W.C. Genes Dev... 1990; 4: 1875-1885Google Scholar, 33Harrod R. Tang Y. Nicot C. Lu H.S. Vassilev A. Nakatani Y. Giam C.Z. Mol. Cell. Biol... 1998; 18: 5052-5061Google Scholar), could activate NF-κB and induce Bcl-xL mRNA expression. Although Tax M22 increased luciferase activity in an HTLV-I LTR-luciferase assay, it failed to increase of NF-κB activity and Bcl-xL expression in both PBT and JPX-9 cells (Fig. 2B). Tax M22 also had a diminished ability to down-regulate PTEN and SHIP-1 mRNA expression as compared with wild-type Tax (Fig. 2C). These results suggested that the NF-κB activation motif in Tax was essential for the down-regulation of PIP3 phosphatase mRNA expression by Tax. Tax-mediated Down-regulation of PIP3-phosphatase mRNA Expression Is Involved in NF-κB Activation—Based on the importance of the Tax NF-κB activation motif, we next asked whether the activation of NF-κB affected the PTEN and SHIP-1 mRNA expression in PBT and JPX-9 cells. The expression of the p65 subunit of NF-κB, which mimics activated NF-κB, significantly increased Bcl-xL expression and NF-κB-Luc activity as described previously (Fig. 3A) (34Chen C. Edelstein L.C. Gelinas C. Mol. Cell. Biol... 2000; 20: 2687-2695Google Scholar, 35Higashitsuji H. Higashitsuji H. Nagao T. Nonoguchi K. Fujii S. Itoh K. Fujita J. Cancer Cell.. 2002; 2: 335-346Google Scholar). We found that p65 drastically decreased both PTEN and SHIP-1 mRNA expression in activated PBT and JPX-9 cells (Fig. 3B). We also examined the effects of a dominant-negative form of IκB (IκBΔN) (36Sun S. Elwood J. Greene W.C. Mol. Cell. Biol... 1996; 16: 1058-1065Google Scholar), which is resistant to phosphorylation and degradation on the down-regulation of PTEN and SHIP-1 by Tax. The expression of IκBΔN reduced not only NF-κB-Luc activity but also the expression of Bcl-xL mRNA, which is normally induced by Tax expression (Fig. 3C). IκBΔN blocked Tax-induced down-regulation of phosphatase mRNA expression (Fig. 3D). These findings indicate that Tax down-regulates expression of both PTEN and SHIP-1 mRNAs through the NF-κB pathway. NF-κB Reduces PIP3 Phosphatase mRNA Expression by Sequestering p300—We further investigated the possibility that the underlying mechanism for Tax-mediated down-regulation of PTEN and SHIP-1 mRNA expression involved the direct interaction between p65 and p300. Using highly p300-expressing cells isolated from p300 wild-type