Transforming Growth Factor-β Directly Induces p53-up-regulated Modulator of Apoptosis (PUMA) during the Rapid Induction of Apoptosis in Myc-driven B-cell Lymphomas^*

Cell Lines and Reagents

BL cell lines were maintained in RPMI 1640 (Invitrogen) supplemented with 5–10% (v/v) heat-inactivated FCS, 2 mm glutamine, and antibiotics. Murine Eμ-Myc lymphoma lines were generated as detailed previously (27) and maintained in DMEM (Invitrogen) supplemented with 10% heat-inactivated FCS, 2 mm glutamine (Invitrogen), 1 mm pyruvate (Invitrogen), 50 μm β-mercaptoethanol (Sigma), 200 μm asparagine (Sigma), and antibiotics. Analyses were performed on four cell lines derived from four independent Eμ-Myc lymphomas isolated from Puma^+/+ mice and on four cell lines derived from four independent Eμ-Myc lymphomas isolated from Puma^−/− mice. The cells were treated as required with 1–5 ng/ml TGF-β1 (Peprotech), 50 μm Z-VAD.FMK (Calbiochem), 5 μg/ml etoposide (Sigma), or 10 μm SB-431542 (Tocris). Protein synthesis inhibition was carried out by preincubation for 2 h with 50 μg/ml cycloheximide and 100 μm anisomycin (Sigma). Inhibition of transcription was carried out by pretreatment of cells for 1 h with 2.5 μg/ml actinomycin D (Sigma). Inhibition of c-Myc activity was carried out by treatment of cells with 25 μm of the c-Myc selective inhibitor 10058-F4 (5-[(4-ethylphenyl) methylene]-2-thioxo-4-thiazolidinone) (Sigma).

Transient Transfection of CA46 BL Cells

5 × 10⁶ CA46 BL cells were transfected in triplicate with 3 μg of firefly luciferase reporter plasmid and 1 μg of Renilla luciferase or β-galactosidase expression plasmids as internal controls for transfection efficiency using an Amaxa nucleofector (solution T, program A-23) as recommended by the manufacturer. Treatment with SB-431542 (10 μm) or solvent vehicle was carried out after transfection if required. Transfected cells were incubated overnight before being treated with TGF-β (5 ng/ml) for 6 h. The ratio of firefly/Renilla luciferase activity was determined using the dual luciferase assay system (Promega) and expressed as the means ± S.D. ratio relative to untreated control samples. The Smad4 shRNA plasmid (TRC 40028) was obtained from Open Biosystems.

Isolation of Centroblasts

CD77+ve cells were purified as described previously (3). Following isolation, the cells were incubated with or without TGF-β in the presence of z-VAD-fmk.

Immunoblotting and Antibodies

Radioimmune precipitation assay lysates were analyzed by SDS-PAGE. Antibodies used in Western blotting were mouse monoclonals against PARP (BD Biosciences); Smad2/3 (BD Biosciences); actin (Sigma); rabbit polyclonal antibodies recognizing phospho-Smad 2 (Ser-465/476), phospho-Smad3 (Ser-433/435), and PUMA (Cell Signaling/Enzo Life Sciences); and a rabbit monoclonal against Tubulin (Cell Signaling). Secondary HRP-conjugated antibodies were obtained from Dako/GE Biosciences. Bound immunocomplexes were detected by enhanced chemiluminescence (ECL; Amersham Biosciences) or Immobilon western chemiluminescent substrate (Millipore).

Analysis of Apoptosis by Flow Cytometry

The cells were fixed in 80% ethanol, labeled with propidium iodide and analyzed by flow cytometry for less than 2 n DNA content. Murine lymphoma lines were stained with FITC-conjugated annexin V (1 μg/ml, kindly provided by Dr. P. Duriez) and propidium iodide (5 μg/ml) in binding buffer (10 mm HEPES, pH 7.4, 140 mm NaCl, 2.5 mm CaCl₂) and analyzed by flow cytometry.

Lentiviral Infection of BL Cell Lines

HEK293FT cells were co-transfected with lentiviral packaging plasmids and pLKO.1-based shRNA constructs (TRC 33610 or TRC 33613; Open Biosystems, termed sh1 and sh2). Lentivirus-containing supernatants were collected, filtered, pooled, and used to infect BL cells. After 48 h, the cells were selected in 0.4 μg/ml puromycin. The resulting stably transduced BL30 cells were routinely passaged in growth medium (RPMI 1640 plus 20% FCS) containing 0.6 μg/ml puromycin. Experiments to analyze TGF-β-induced apoptosis were carried out in the absence of selection medium.

Retroviral Infection of Murine Eμ-Myc Lymphoma Lines

Ecotropic Phoenix cells were treated with 5 μm chloroquine 30 min prior to calcium: phosphate precipitation-mediated transfection with the pMIH vector (modified pMIG, GFP cassette exchanged for hygromycin resistance) encoding a human BCL-2 transgene, or vector alone, alongside the helper vector pCL.Eco. After 48 h, virus-containing supernatants were removed and filtered, and Polybrene was added to 4 μg/ml. Eμ-Myc lymphomas were plated at 6 × 10⁵ cells/well in a 12-well plate, and 1.5 ml of virus-containing medium was applied. The plates were centrifuged at 480 × g at 37 °C for 45 min. Selection was then carried out using 250 μg/ml hygromycin for 7 days. Subsequently, BCL-2 expression levels were analyzed by flow cytometry and Western blot.

qRT-PCR

Total cell RNA was isolated using TRIzol as recommended by the manufacturer. cDNA and qRT-PCRs were prepared using SYBR green two-step qRT-PCR kits (Finnzymes) and specific primers for each gene (Qiagen). Amplified products were analyzed by Chromo4 continuous fluorescence detector and Opticon Monitor3 software. The relative amount of RNA for each gene is expressed after normalization to the amount of 18 S rRNA in each sample. RNA isolated from murine lymphomas was converted to cDNA using a first strand synthesis RT-PCR kit (Invitrogen). Quantitative PCRs were performed using qPCR Supermix-UND (Invitrogen) and TaqMan gene-specific primers (Applied Biosystems). Amplification and analysis was achieved via use of a C1000 thermal cycler fitted with a CFX96 fluorescence detector (Bio-Rad) and CFX96 manager software (Bio-Rad).

ChIP Assay

2 × 10⁷ CA46 cells were treated with 1 ng/ml TGF-β for 1 h and fixed in 1% formaldehyde for 15 min. The formaldehyde was quenched with glycine, the cells were washed in PBS, and nuclear extracts were prepared. The extracts were sonicated to fragment the DNA and incubated overnight with 20 ng of goat anti-Smad3 or 4 (Santa Cruz) or isotype-matched control antibodies. Immune complexes were captured using anti-goat IgG-coated agarose beads, the samples were washed, and the DNA was eluted off the beads. DNA cross-links were reversed by incubation at 65 °C in 0.3 m NaCl, and the samples were then digested with proteinase K. DNA was extracted using phenol/chloroform and ethanol-precipitated. PCRs using specific primers for human PUMA (5′-AGACTTGCTTGAACCCAGGA-3′ and 5′-AATCGCCTCACCAGAGTGAC-3′) were performed for 35–50 cycles, and the PCR products were analyzed on 2% agarose gels stained with SYBR Green.

TGF-β Induces the BH3-only Activator PUMA

We previously showed that TGF-β treatment of BL cells results in the transcriptional regulation of members of the BCL-2 family of apoptosis regulators including BIK and BCL-X_L (3). Analysis of other BH3-only proteins that might contribute to the apoptotic program induced by TGF-β was carried out on subsequent TGF-β treatment time courses (Fig. 1). Apoptosis was evident, as detected by Western blotting for cleavage of the caspase-3 substrate PARP, as early as 6–8 h after TGF-β addition (Fig. 1A). During this period, the induction of BIK mRNA was confirmed; however, we also detected a transient increase in transcripts of the apoptosis activator PUMA (Fig. 1B). There was no concurrent increase in expression levels of the apoptosis sensitizers, NOXA, BAD, HRK, or BMF. The induction of PUMA mRNA in response to exogenous TGF-β addition was confirmed across a panel of BL cell lines (Fig. 2A) and at the level of protein expression (Fig. 2B). The Western blot analysis shown in Fig. 2B indicates that the α isoform but not the β isoform of PUMA was induced in response to TGF-β signaling.

BLs are derived from centroblasts within the germinal centers of secondary lymphoid tissue. By isolating CD77-positive centroblasts from human tonsil tissue (as described in Ref. 3), we were able to demonstrate a small but consistent increase in PUMA mRNA expression in TGF-β treated cells relative to untreated controls. No comparable increase in either NOXA or HRK mRNA expression was evident (Fig. 2C). Increased abundance of PUMA RNA transcripts in the presence of TGF-β was also evident throughout a 24-h time course of treatment of CD77-positive primary cells (Fig. 2D). PUMA can therefore be induced in response to TGF-β in both BL cell lines and in the cell of origin isolated from primary tissue. Induction of PUMA mRNA in response to exogenous TGF-β addition could also be detected in the pro-B-cell line RS4;11, but not in non-B-cell lineages including mouse embryo fibroblasts and HaCaT epithelial cells (data not shown).

PUMA Is a Direct Target Gene of TGF-β Signaling in Human B-cells

We next investigated the mechanism of TGF-β-mediated regulation of PUMA expression first by testing whether PUMA is an immediate early target of TGF-β signaling. BL cells were pretreated with cycloheximide and anisomycin and were subsequently treated with exogenous TGF-β for the times indicated. Induction of PUMA occurred even in the absence of de novo protein synthesis (Fig. 3A), but blocking transcription with actinomycin D resulted in the complete ablation of PUMA mRNA induction (Fig. 3B). c-Myc overexpression is characteristic of BL cells, and c-Myc itself is known to repress PUMA transcription following growth factor stimulation (28). We tested the effect of c-Myc overexpression on TGF-β-induced activation of PUMA by pretreating BL cells with the c-Myc inhibitor 10058-F4 at concentrations previously shown to induce BL cell death (29). Inhibition of c-Myc modestly increased basal PUMA transcription (especially in L3055 cells) but did not significantly affect PUMA induction in response to TGF-β (Fig. 3C). Our data indicate that PUMA is a direct target of TGF-β signaling and, as such, implies that PUMA transcription is likely to be regulated by Smad complexes binding to a cognate binding element within the gene promoter. Smad-binding regions (SBRs) frequently contain two copies of the Smad-binding element (SBE) sequence 5′-GTCT-3′ or its reverse complement 5′-AGAC-3′ (30). We identified a potential SBR (5′-agactctgtct-3′) (Fig. 3D) ∼1.9 kb upstream of the PUMA transcription start site, contained within a region of the promoter bearing striking homology to the SBR we previously identified within the human BIK promoter (Fig. 3D and Ref. 3). ChIP analysis using primers spanning this region demonstrated TGF-β-dependent binding of Smads 3 and 4 to the endogenous PUMA promoter in vivo (Fig. 3E).

We next analyzed the activity of a luciferase promoter reporter construct containing ∼2 kb of the 5′ region upstream of the PUMA transcription start site (31). The PUMA promoter construct was constitutively active in CA46 BL cells and could not be further induced by the addition of exogenous TGF-β (Fig. 4A), suggesting that the endogenous level of TGF-β signaling in these cells may be sufficient for maximal activity of the isolated promoter region. We therefore tested whether inhibition of basal TGF-β signaling using 10 μm of the ALK5 inhibitor SB-431542 would affect PUMA promoter activity (Fig. 4B). We observed an approximate 50% decrease in luciferase activity following SB-431542 treatment, indicating that TGF-β signaling is required for maximal promoter activity.

To examine the activity of the potential Smad-binding region more directly, we generated firefly luciferase reporter constructs containing concatemerized (2×) versions of wild type and mutant sequences shown in Fig. 4C. Consistent with the 2-kb promoter fragment, the 2× wild type SBE sequence was active in CA46 cells. This activity was dependent on the consensus SBEs because mutation of the sequence agactctgtct to atattctatat reduced reporter activity to near basal levels (Fig. 4D). Inhibition of endogenous TGF-β signaling also reduced activity of the 2× wild type SBE reporter (Fig. 4E).

We next transiently transfected a shRNA construct targeting Smad4 to determine whether Smad4 is required for PUMA promoter activity. Because BL cells are renowned for their low transfection rate, we monitored efficacy of Smad4 knockdown by inhibition of the Smad4 dependent CAGA₁₂-luciferase reporter response to TGF-β addition (Fig. 4F). We were able to achieve a partial inhibition of CAGA₁₂ activity and likewise observed a small but significant decrease in PUMA activity (Fig. 4G). Taken together, these findings indicate that regulation of the PUMA promoter by TGF-β signaling is most likely dependent on Smad binding at the SBR at positions −1923 to −1885 and that proper regulation of the endogenous gene depends on the correct repressive chromatin context.

PUMA Knockdown Retards TGF-β-induced Apoptosis

To determine the functional consequence of PUMA induction by TGF-β, we used lentiviral transduction of shRNA constructs to stably knockdown PUMA in BL30 cells. Transducing BL30 cells with a combination of two commercially available shRNA constructs (shRNA1 + 2) reduced the basal and TGF-β-inducible PUMA levels to below the background levels observed in the control line (Fig. 5A). Two PUMA knockdown stable cell pools (shRNA1 + 2#1 and shRNA1 + 2#2) were analyzed for apoptosis induction by TGF-β using propidium iodide staining and flow cytometry. Knockdown of PUMA resulted in a significant reduction in the amount of apoptosis induction by TGF-β after 24 h of treatment (Fig. 5B). We noted, however, that at later time points (48 h) there was little discernable difference between apoptosis induction in control versus PUMA knockdown cell lines (data not shown). We therefore carried out a more detailed kinetic analysis of early apoptosis induction using PARP cleavage as a measure (Fig. 5C, top panel). The Western blots revealed that although all lines retained equivalent TGF-β signaling, shown by equal levels of phosphorylation of the receptor regulated Smad2, the apoptotic response in PUMA knockdown lines was retarded when compared with the control line, 22 and 18% PARP cleavage in the knockdown lines after 8 h of treatment compared with 50% (summarized in the graph shown in Fig. 5C, bottom panel).

TGF-β Induces Apoptosis and PUMA Expression in Eμ-Myc Murine Lymphoma Lines

Given that PUMA expression levels in the stable knockdown cell lines were reduced to below the basal levels detected in untreated controls (Fig. 5A), the lack of any significant effect on the percentage of apoptotic cells at later time points is unlikely to be a consequence of the incomplete inhibition of PUMA induction in response to TGF-β. However, to exclude this possibility, we extended our study to compare wild type and Puma null murine B-lymphoma cells. Eμ-Myc murine lymphoma lines are a model of BL. We first determined whether isolated wild type Eμ-Myc lymphoma cells undergo apoptosis in response to TGF-β addition in vitro. Annexin V staining of Eμ-Myc cells revealed TGF-β-induced cell death after 24 h of treatment (Fig. 6A). Apoptosis was confirmed by cleavage of PARP in TGF-β-treated wild type lymphoma cells (Fig. 6B). Cell death in response to TGF-β was dependent on the TGF-β type I receptor ALK5 because pretreatment of lymphoma cells with SB-431542 inhibited death induced by TGF-β but had no effect on the cellular response to the DNA damage-inducing agent etoposide (Fig. 6, C and D). Apoptosis was inhibited by overexpression of BCL-2 in murine lymphoma lines, indicating the involvement of the intrinsic apoptosis pathway (Fig. 6, E and F). In addition, TGF-β treatment resulted in induction of Puma shown by quantitative RT-PCR (Fig. 6G) and by Western blot (Fig. 6H). These data indicate that wild type murine Eμ-Myc lymphoma cells mimic the response of human BL cell lines to TGF-β and undergo ALK5-dependent intrinsic apoptosis involving PUMA induction.

PUMA Is Required for Efficient Apoptosis Induction in Eμ-Myc Lymohoma Lines

shRNA knockdown of PUMA in human BL cell lines delayed the onset of apoptosis in response to TGF-β (Fig. 5C). We observed a similar correlation in the murine Eμ-Myc line 16, which had slower Puma RNA induction after TGF-β addition (Fig. 7A) and lower levels of apoptosis by 12 h of TGF-β treatment (Fig. 7B). However, to definitively investigate the role of PUMA in this response, we compared the response of wild type Eμ-Myc cell lines with Puma null lines (Puma^−/−) to TGF-β. All lines responded to TGF-β because they showed similar levels of Smad2 phosphorylation following exogenous TGF-β addition (Fig. 7C). When the effects of TGF-β treatment were directly compared in wild type versus Puma null cells, there were significantly fewer apoptotic Puma null cells than apoptotic wild type cells at early time points following exposure to TGF-β (Fig. 7D) but not at later time points (Fig. 7E), thus confirming the data obtained in human BL lines. PUMA is therefore selectively required for efficient, rapid TGF-β-induced apoptosis.