Downregulation of proapoptotic Bim augments IL-2-independent T-cell transformation by human T-cell leukemia virus type-1 Tax

Human T-cell leukemia virus type 1 (HTLV-1), an etiological agent of adult T-cell leukemia, immortalizes and transforms primary human T cells in vitro in both an interleukin (IL)-2-dependent and IL-2-independent manner. Expression of the HTLV-1 oncoprotein Tax transforms the growth of the mouse T-cell line CTLL-2 from being IL-2-dependent to IL-2-independent. Withdrawal of IL-2 from normal activated T cells induces apoptosis, which is mediated through the inducible expression of several proapoptotic proteins, including Bim. In this study, we found that Tax protects IL-2-depleted T cells against Bim-induced apoptosis. Withdrawal of IL-2 from CTLL-2 cells induced a prominent increase in the level of Bim protein in CTLL-2 cells, but not in Tax-transformed CTLL-2 cells. This inhibition of Bim in Tax-transformed CTLL-2 cells was mediated by two mechanisms: downregulation of Bim mRNA and posttranscriptional reduction of Bim protein. Transient expression of Tax in CTLL-2 cells also inhibited IL-2 depletion–induced expression of Bim, however, this decrease in Bim protein expression was not due to downregulation of Bim mRNA, thus indicating that Bim mRNA downregulation in Tax-transformed CTLL-2 occurs only after long-term expression of Tax. Transient expression of Tax in CTLL-2 cells also induced Erk activation, however, this was not involved in the reduction of Bim protein. Knockdown of Bim expression in CTLL-2 cells augmented Tax-induced IL-2-independent transformation. HTLV-1 infection of human T cells also reduced their levels of Bim protein, and restoring Bim expression in HTLV-1-infected cells reduced their proliferation by inducing apoptosis. Taken together, these results indicate that Tax-induced downregulation of Bim in HTLV-1-infected T cells promotes their IL-2-independent growth, thereby supporting the persistence of HTLV-1 infection in vivo.


Introduction
Adult T-cell leukemia (ATL) is a highly aggressive human leukemia characterized by the clonal proliferation of mature T cells and is initiated by an infection with human T-cell leukemia virus type 1 (HTLV-1) [1,2]. HTLV-1 transmission mainly occurs from mothers to their infants through breast milk. Approximately 3-5% of HTLV-1 infections develop into ATL, at an average age of 60 years [3]. During asymptomatic periods, host immunity suppresses the outgrowth of HTLV-1-infected cells. However, two events, deterioration of host immunity and emergence of a malignant clone with genetic and epigenetic alterations, overcome suppression against HTLV-1infected T cells, resulting in the development of ATL [4,5].
HTLV-1 immortalizes primary human T cells in vitro, and subsequently some cells acquire an interleukin-2 (IL-2)-independent growth phenotype [6]. This HTLV-1induced immortalization and transformation event is essential for establishing a lifelong persistent infection. In addition to structural genes, HTLV-1 encodes two oncoproteins, Tax and HBZ [1]. Of the two, Tax is essential for HTLV-1-mediated IL-2-dependent immortalization of T cells. For example, mutation of the tax gene in a recombinant HTLV-1 strain abolishes its immortalization activity in T cells [7]. Moreover, Tax alone, without any other viral genes, can immortalize T cells in vitro [8,9]. In addition to IL-2-dependent immortalization, Tax may also play a role in the IL-2-independent transformation of T cells by HTLV-1. For instance, transduction of the tax gene into the mouse IL-2-dependent T-cell line CTLL-2 confers IL-2-independent growth [10]. Tax has been reported to repress the proapoptotic Bcl-2 family protein Bax and induce the antiapoptotic proteins Bcl-xL and Bfl-1 [11][12][13]. However, how Tax induces the IL-2-independent growth transformation in T cells has not been fully elucidated.
Upon depletion of IL-2, activated normal T cells initiate apoptosis through the induction of several proapoptotic genes, including Bim and Fas ligand [14]. Bim is a proapoptotic BH3-only protein, which binds to all members of the antiapoptotic Bcl-2 family [15]. In this study, we examined how Tax prevents Bim-induced apoptosis of T cells after IL-2 depletion. We present evidence that downregulation of Bim in T cells plays a crucial role in the IL-2-independent growth of HTLV-1-infected T cells, including ATL-derived cells.

Lentiviruses
Recombinant lentiviruses were generated by transfecting each lentiviral vector together with pCAG-HIVgp and pCMV-VSV-G-RSV-Rev (provided by Dr. H. Miyoshi, RIKEN Tsukuba Institute) into 293T cells by lipofection using FuGENE HD (Promega, Madison, WI). Since transfection of the BimEL-expressing lentiviral vector into 293T cells induced cell death, the pSVBT plasmid expressing the human antiapoptotic protein Bcl-2 (provided by Dr. Y. Tsujimoto at Osaka University) was cotransfected into 293T cells. The supernatant of 293T cells containing the lentiviruses was used to infect CTLL-2, TL-OmI, and ST1 cells (2-4 9 10 5 ) in a final volume of 1 mL of RPMI/10% FBS containing 8 lg/mL polybrene, supplemented with 0.5 nmol/L IL-2 for CTLL-2 cells. The efficiency of these infections was augmented by centrifugation of the infected cells with the viruses (800g at 32°C for 1 h) as described previously [25]. To establish stably infected CTLL-2 cell lines, infected cells were cultured in selection medium containing 28 lg/mL blasticidin or 2 lg/mL puromycin for 1-2 weeks.

Real-time PCR
Total RNA was isolated from CTLL-2 or CTLL-2/Tax cells using the RNAiso reagent (Takara Bio, Otsu, Japan). Five hundred nanograms of total RNA was reverse-transcribed using the ExScript RT reagent kit (Takara Bio). To quantify the amount of Bim RNA, realtime PCR based on SYBR green fluorescence was performed using SYBR Premix Ex Taq polymerase and the Thermal Cycler Dice real-time system (Takara Bio). Primers specific for mouse Bim and glyceraldehyde-3phosphate dehydrogenase (GAPDH) RNAs were purchased from Takara Bio.

Transformation assay
The IL-2-independent transformation assay was performed as described previously [16]. Briefly, CTLL-2 cells were infected with the Tax or Tax225-232 lentivirus, and the cells were cultured in 96-well plates (0.1 9 10 4 , 0.3 9 10 4 , and 1 9 10 4 cells per well) without IL-2 for 4 weeks. The number of wells containing outgrowing cells was counted under a light microscope.

Cell death assays
Cell viability was assessed by staining with trypan blue under a light microscope. To monitor for apoptosis, cells were stained with 5 lg/mL of Hoechst 33342 for 30 min at 37°C and examined with an inverted fluorescence microscope (Keyence, Osaka, Japan).

Statistical analysis
Statistical analysis was performed using GraphPad Prism 5 software (GraphPad Software Inc., San Diego, CA).

Bim downregulation in Tax-transformed CTLL-2 cells
CTLL-2 is a mouse T-cell line that requires IL-2 for continuous growth. Since depletion of IL-2 from CTLL-2 cultures induces apoptosis, we first examined whether IL-2 withdrawal from CTLL-2 cells induces the expression of the proapoptotic protein, Bim. Western blot analysis showed that IL-2 withdrawal from CTLL-2 cells for 18 h markedly induced the expression of BimEL, BimL, and BimS ( Fig. 1A), which are proteins encoded by the three alternatively spliced variants of the identical Bim transcript. All three isoforms have a proapoptotic function, with BimS being the most potent [27]. This observation suggests that Bim is one factor responsible for IL-2 depletion-induced apoptosis of CTLL-2 cells.
We have previously shown that Tax transforms the growth of CTLL-2 cells from being IL-2-dependent to IL-2-independent [10]. Therefore, we next examined how Tax inactivates Bim in IL-2-depleted CTLL-2 cells. We found that the amount of Bim proteins in Tax-transformed CTLL-2 cells in the absence of IL-2 was much lower than that in IL-2-depleted parental CTLL-2 cells, and was even lower than that in IL-2-supplemented CTLL-2 cells (Fig. 1A). In addition, four independently established Tax-transformed IL-2-independent CTLL-2 clones displayed a reduced amount of Bim protein in the absence of IL-2 (Fig. 1B). These results suggest that Tax prevents IL-2 depletion-induced apoptosis of CTLL-2 cells partly through reducing the level of Bim protein.
We next examined whether Tax also represses Bim at the RNA level in CTLL-2 cells. Real-time PCR analysis showed that Bim mRNA in CTLL-2 cells was greatly induced by IL-2 deprivation, but no induction of Bim mRNA was observed in Tax-transformed CTLL-2 cells cultured in the absence of IL-2 (Fig. 1C). These results suggest that Tax-induced IL-2-independent transformation of CTLL-2 cells is due to downregulation of Bim at both the protein and the mRNA levels.
To further establish that Tax reduces the expression of Bim in CTLL-2 cells, we transiently expressed Tax in CTLL-2 cells by infection with a Tax-expressing lentivirus. The Tax virus-infected cells were cultured in the presence of IL-2 for 48 h and further cultured without IL-2 for 18 h. Transient expression of Tax in CTLL-2 cells reduced the amount of BimEL and BimL protein, with the reduction of BimEL being greater than that of BimL ( Fig. 2A). It should be noted that BimEL, but not BimL, has three Erk phosphorylation sites, serines 55, 65, and 73, which control Erk-induced degradation of Bim [35]. The greater amount of p-Erk in Tax-expressing CTLL-2 cells than in control CTLL-2 cells and the slightly slower SDS-PAGE gel migration of BimEL, but not BimL, in Tax-expressing CTLL-2 cells ( Fig. 2A) are consistent with Tax-induced Erk-dependent phosphorylation of BimEL. Collectively, these results suggest that transient expression of Tax activates Erk to phosphorylate BimEL, thereby inducing the degradation of BimEL. Consistent with this, transient expression of Tax in CTLL-2 cells minimally affected the level of Bim mRNA (Fig. 2B). Therefore, the observed downregulation of Bim mRNA in Tax-transformed IL-2-independent CTLL-2 cells is either a consequence of long-term expression of Tax or due to selection for Bim low-expressing cells during the Tax-induced IL-2-independent transformation process of CTLL-2 cells.
To further examine the finding that Tax reduces Bim protein level through Erk activation, CTLL-2 cells were infected with lentivirus coexpressing Tax and blasticidin-S-deaminase, and the cells were cultured with IL-2 and blasticidin for 7 days. These cells were treated with the MEK inhibitor U0126 in the absence of IL-2, and the amount of Bim in these cells was examined ( Fig. 2C and D). While Tax reduced the amount of BimEL protein in CTLL-2 cells and U0126 almost completely blocked Erk activation by Tax, U0126 did not significantly affect the reduction of BimEL, indicating that Tax reduces Bim protein by an Erk-independent mechanism.
Next, we characterized various Tax mutants for their Bim-reducing activities (Fig. 2E). To do so, CTLL-2 cells were infected with lentivirus coexpressing Tax and blasticidin-S-deaminase, cultured with IL-2 and blasticidin for 7 days, and examined for Bim and p-Erk expression after IL-2 withdrawal for 18 h. Wild-type Tax (WT-Tax) reduced the expression of BimEL but not BimL in CTLL-2 cells. Among the Tax mutants, TaxDC and M22 did not induce a reduction in Bim expression. Since TaxDC lacks a PDZ domain-binding motif (PBM) [22] and M22 does not activate NF-jB-dependent transcription [23], these results suggest that functions mediated by the PBM and NF-jB signaling pathways are both required for Taxinduced Bim reduction. It should be noted, however, that the amount of TaxDC and M22-mutant proteins in CTLL-2 cells was lower than that of WT-Tax. Thus, further analysis is required to confirm the above conclusion. In contrast, Tax703 and Tax225-232 reduced Bim expression equivalent to WT-Tax in CTLL-2 cells. Since Tax703 and Tax225-232 are defective for CREB activation [23] and NF-jB2-dependent transcriptional activation [21], respectively, our results indicate that the activation of both CREB and NF-jB2 pathways by Tax are dispensable for the Tax-induced reduction of Bim.

Knockdown of Bim augments the transforming activity of Tax in CTLL-2 cells
To investigate whether Bim downregulation by Tax is involved in the transformation of CTLL-2 cells, we established Bim-knockdown CTLL-2 cells by infection of lentivirus expressing shRNA targeting Bim. BimEL and BimL were efficiently knocked down in these cells, but the levels of other Bcl-2 family proteins, Bax and Bcl-xL, were not affected (Fig. 3A). Bim knockdown did not induce IL-2-independent growth transformation of CTLL-2 cells without Tax (data not shown). Next, Bimknockdown and control CTLL-2 cells were infected with lentivirus encoding WT-Tax or a Tax mutant (Tax225-232) defective for NF-jB2 activation, and the cells were cultured in the absence of IL-2 for 4 weeks. Tax efficiently induced IL-2-independent transformation in CTLL-2 cells, and the transforming activity was augmented by Bim knockdown (Fig. 3B). Consistent with our previous report [21], Tax225-232 had a lower transformation capacity than WT-Tax in the control cells, but its transforming activity in CTLL-2 was also greatly improved by Bim knockdown. The expression levels of WT and mutant Tax proteins 48 h after infection with their corresponding lentiviral vectors were almost equivalent (Fig. 3B). Taken together, these results indicate that Bim induction by IL-2 deprivation in CTLL-2 cells inhibits their transformation by WT-Tax and Tax225-232, and that Bim downregulation by Tax is an important activity for the cells' acquisition of IL-2 independence. In addition, these results suggest that the lower transforming activity of Tax225-232 relative to WT-Tax is mediated by the mechanism distinct from the reduction of Bim.
We next compared the role of Tax expression and Bim-knockdown in CTLL-2 cells during the initial phase of CTLL-2 cell transformation. CTLL-2 cells transduced with Tax-lentivirus in the presence of IL-2 and Bimknockdown CTLL-2 cells were cultured in the absence of IL-2 (Fig. 3C, right). Tax expression in CTLL-2 cells did not confer a survival advantage compared with Bimknockdown after 3 days, however, at day 4 Tax-expressing cells, but not Bim-knockdown cells, started to grow. These results suggest that BimEL downregulation by Tax in CTLL-2 is not enough to inhibit apoptosis of CTLL-2 after IL-2 withdrawal. Consistently, Bim knockdown only partially protected CTLL-2 cells from IL-2-depletioninduced apoptosis (Fig. 3C, left).

Downregulation of Bim in HTLV-1-infected T-cell lines
We next investigated the status of Bim protein in HTLV-1-transformed IL-2-independent T-cell lines and in T-cell lines that originated from ATL patients. Western blot analysis showed that five of the six HTLV-1transformed T-cell lines (all except HUT102) expressed a reduced amount of Bim protein relative to uninfected T-cell lines and PHA-activated peripheral blood mononuclear cells (PHA-PBMC) (Fig. 4A). Since HTLV-1transformed cells express less BimL and BimS than uninfected T-cell lines, the corresponding Bim mRNAs are presumed to be downregulated in these HTLV-1transformed cell lines. These results suggest that downregulation of Bim protein is a factor required for HTLV-1-mediated IL-2-independent transformation of human T cells. In addition, four of the five ATL cell lines tested (all except MT-1) expressed a reduced amount of Bim protein relative to PHA-PBMC (Fig. 4B). It should be noted that MT-1 expressed a relatively high level of p-Erk. Furthermore, the BimEL detected in MT-1 cells had a slightly higher molecular weight than that in ST1 cells, suggesting that it may be inactivated by posttranslational modifications such as phosphorylation. Collectively, these results suggest that Bim is inactivated in ATL cells. Of the five ATL cell lines, only KOB cells expressed a detectable amount of Tax. Thus, the downregulation of Bim protein in ATLderived cell lines is mostly Tax-independent, and another viral protein or a host factor appears to control the amount of Bim in ATL cells.

Re-expression of Bim in ATL cell lines induces apoptosis
To establish whether the downregulation of Bim in ATL cell lines plays a role in their aberrant growth properties, we re-expressed Bim in Bim-negative ATL cell lines. TL-OmI and ST1 cells were infected with the EGFP-or BimEL-expressing lentivirus, and 48 h after infection cell viability was determined by trypan blue staining. BimEL was detected in BimEL-transduced ST1 cells but not in control cells (Fig. 5A). Furthermore, BimEL re-expression in the two ATL cell lines significantly induced cell death compared with control expression of EGFP (Fig. 5B). Hoechst 33342 staining revealed condensed chromatin in BimEL-expressing ST1 cells, indicating that the observed cell death was induced by an apoptotic mechanism (Fig. 5C). These results demonstrate that the downregulation of Bim in ATL cells is required for their aberrant cell growth and survival.

Discussion
HTLV-1 immortalizes and transforms human T cells in both an IL-2-dependent and IL-2-independent manner in vitro, suggesting that HTLV-1-infected cells in vivo grow at both high-IL-2 and low-IL-2 conditions [36]. In this study, we found that Tax attenuates the induction of Bim protein in the T-cell line CTLL-2 after IL-2 depletion, and that this reduction promotes the Tax-induced IL-2-independent transformation of CTLL-2 cells. Moreover, HTLV-1-transformed IL-2-independent human Tcell lines generally displayed a low level of Bim protein relative to control T-cell lines. These results suggest that Tax-induced Bim reduction plays a role in cell growth in HTLV-1-infected T cells under IL-2-depleted or low-IL-2 conditions. Reduction of Bim in Tax-transformed or Tax-expressing CTLL-2 cells seems to occur by two distinct mechanisms, one being the downregulation of Bim mRNA level. Since transient expression of Tax in CTLL-2 cells did not reduce Bim mRNA, there are two possible mechanisms to explain the downregulation of Bim mRNA in Tax-transformed cells. First, because Tax interacts with several chromatin remodeling factors, such as the histone methyltransferase SUV39H1 [37], Tax might induce the silencing of the Bim gene. Alternatively, only CTLL-2 cells expressing low levels of Bim may be selected during Tax-induced transformation of CTLL-2 cells.
The second mechanism of Bim reduction in CTLL-2 cells is posttranscriptional reduction of Bim protein. Bim is known to be phosphorylated by Erk and degraded in a phosphorylation-dependent manner. Stoppa et al. showed that Tax expression in the human T-cell line Jurkat increased the levels of p-Erk, thereby inhibiting cisplatininduced apoptosis [38]. They further showed that Tax activates Ras, an upstream activator of Erk, suggesting that Tax increases p-Erk by stimulating Ras activity. We also observed that Tax increased the amount of p-Erk present in CTLL-2 cells and that this activation was blocked by the MEK inhibitor U0126, suggesting Erk activation by Tax is mediated through the upstream kinase MEK. However, our data also demonstrated that Erk activation by Tax is not responsible for BimEL degradation. Since Tax has been reported to activate several kinases, including IKKa [39], IKKb [40,41], AKT [42], mTOR [43], TAK1, and JNK [44], there are multiple candidates possibly responsible for BimEL phosphorylation by Tax. Although Bim degradation by Tax was unable to protect CTLL-2 cells from apoptosis after IL-2 withdrawal, such activity would have a critical role in establishing HTLV-1 infection in vivo.
In some HTLV-1 infected cell lines, Erk activation and BimEL phosphorylation did not correlate with the reduction of BimEL protein. It has been previously reported that Erk-dependent BimEL phosphorylation does not induce BimEL protein degradation in chronic lymphoblastic leukemia cells, but instead reduces its binding to the antiapoptotic protein Mcl1, thus contributing to cell survival [45]. It is possible that the same mechanism of BimEL inhibition operates in HTLV-1-infected cells or ATL cell lines.
Intriguingly, the amount of Bim protein was low in ATL-derived T-cell lines, even though they expressed a very small or nearly undetectable amount of Tax. Moreover, re-expression of Bim in ATL cell lines reduced cell growth by inducing apoptosis. These results indicate that the aberrant growth of ATL cells depends on Bim repression. Thus, Bim may be a good target molecule for therapeutics against ATL. For instance, a Bim mimetic, such as ABT-737, might be a good candidate for anti-ATL therapeutics [46].
In addition to Tax, several viral oncoproteins including LMP2A of Epstein-Barr virus and viral interferon regulatory factor 1 of human herpesvirus 8 inhibit Bim function [47,48]. Specifically, LMP2A inhibits Bim-induced anoikis by promoting Erk-dependent Bim degradation in epithelial cells [47]. Thus, Bim is a common target for oncogenic viruses to establish persistent infection. Further investigations to elucidate the mechanism of Bim downregulation in HTLV-1-infected cells will advance the understanding of viral pathogenesis, including HTLV-1-induced ATL.