Smad3 Potentiates Transforming Growth Factor β (TGFβ)-induced Apoptosis and Expression of the BH3-only Protein Bim in WEHI 231 B Lymphocytes*

Transforming growth factor-β (TGFβ) is a potent growth inhibitor and inducer of apoptosis in B lymphocytes and is essential for immune regulation and maintenance of self-tolerance. Here we show that exogenous overexpression of Smad3 potentiates TGFβ-induced apoptosis and expression of the pro-apoptotic protein Bim in WEHI 231 B lymphocytes. Overexpression of dominant-negative forms of Smad3 abrogate these TGFβ-induced responses. We also demonstrate that TGFβ induces Bim protein expression concomitant with its induction of apoptosis in the mouse progenitor B lymphocyte cell line, Ba/F3. Enhanced expression of Bim protein induced by TGFβ is associated with an increased association of Bim with Bcl-2 and a concomitant loss of mitochondrial membrane potential. Furthermore, we find that the anti-apoptotic effect of the pro-survival cytokine CD40 results in the abrogation of TGFβ-mediated Bim induction. Our data provide the first evidence of Bim expression levels that are increased by the addition of a pro-apoptotic cytokine, TGFβ, and also suggest that the TGFβ−specific transcription factor Smad3 plays a role in mediating Bim expression levels and apoptosis.

Transforming growth factor ␤ (TGF␤) 1 and its related factors modulate essential cellular functions ranging from cellular proliferation and differentiation to apoptosis (1)(2)(3)(4). Signaling by TGF␤ is initiated by an oligomeric receptor complex consisting of two types of transmembrane subunits that each possess serine/threonine kinase activity. Binding of ligand to the constitutively active type II receptor (T␤RII) promotes complex formation with the type I receptor (T␤RI/ALK5). Subsequent phosphorylation and activation of T␤RI/ALK5 by T␤RII leads to further propagation of TGF␤ signaling by several signaling cascades, which include the Smads, MAPK, and PI3K (1)(2)(3)(4).
Signaling by TGF␤ through the Smad pathway has been extensively characterized and is considered the canonical pathway. Receptor-regulated Smads (R-Smads), Smad2 and Smad3, are directly phosphorylated and activated by ALK5. Phosphorylation occurs at C-terminal SSXS motifs and promotes the formation of heteromeric complexes with the common mediator Smad, or co-Smad, Smad4. The Smad complexes translocate into the nucleus, where they regulate gene expression by directly interacting with resident DNA-binding proteins and by recruiting co-activators or co-repressors to the promoter (1)(2)(3)(4). Under basal conditions, R-Smads have been shown to be retained in the cytoplasm through their interaction with membrane-anchoring proteins containing FYVE domains, such as SARA (5) and Hgs/Hrs (6), thereby facilitating R-Smad activation by TGF␤ receptors. Recently, we have shown that the adaptor molecule disabled-2 (Dab2) links TGF␤ receptors to Smad proteins (7), presumably in early endocytotic vesicles because of the interaction of Dab2 with the clathrin adaptor molecule AP-2 (8).
In addition to the canonical Smad pathway, TGF␤ has also been reported to signal through components of the MAPK and PI3K/Akt pathways. TGF␤ has been shown to activate extracellular signal-regulated kinase (ERK) (9,10), Jun N-terminal kinase (JNK) (11)(12)(13)(14), p38 mitogen-activated protein kinase (p38) (15,16), and PI3K/AKT (17). The TGF␤ responses regulated by these kinases are varied ranging from reporter construct transactivation to regulation of cellular proliferation and apoptosis. The kinetics of these responses also vary in magnitude and duration, and there are reports suggesting that members of the Rho family of small GTPases may directly couple activated TGF␤ receptors to these signaling pathways (11, 18 -20) or that activation of these pathways may be indirect, possibly resulting from Smad-dependent transcriptional responses.
TGF␤ exerts both pro-apoptotic and anti-apoptotic effects depending on the cell type or cellular context. Pro-apoptotic responses have been demonstrated in prostate epithelium (21,22), hepatocyte and hepatoma cell lines (23)(24)(25), hematopoietic cells (26), and in B lymphocytes (27)(28)(29). The molecular mechanisms mediating the pro-apoptotic effects of TGF␤ are not completely understood and appear to be cell type-dependent. Recently, it has been shown that Daxx, a Fas-receptor-associated protein that activates the JNK pathway, interacts directly with T␤RII and couples TGF␤ signaling to the apoptotic machinery in AML12 hepatocytes (30). In the Hep3B hepatoma cell line, TGF␤ has been shown to induce Smad-dependent expression of the death-associated protein kinase (DAP-kinase), a calcium/calmodulin-regulated serine/threonine kinase previously implicated in several apoptotic responses (31). Another study reports that ARTS (apoptotic protein in the TGF␤ signaling pathway), a septin-like protein, translocates from the mitochondria to the nucleus in response to TGF␤ treatment of the prostatic epithelial cell line NRP-154 (32). The Bcl-2 family of proteins has also been implicated as mediators of TGF␤induced apoptosis. Early studies in the WEHI 231 B lymphocyte cell line demonstrated that stable overexpression of Bcl-X L abrogated TGF␤-mediated apoptosis (27). More recently, it was shown in the FaO rat hepatoma cell line that TGF␤ does not effect the expression levels of many members of the Bcl-2 family but did induce the caspase-dependent cleavage of BAD, a pro-apoptotic Bcl-2 family member (33). Overexpression of Smad3 in these cells was shown to promote the caspase 3-mediated cleavage of BAD and apoptosis, whereas antisense Smad3 cDNA blocked TGF␤-mediated apoptosis and BAD cleavage (33).
We have recently shown that TGF␤-mediated apoptosis in WEHI 231 B lymphocytes can be blocked by overexpression of the inhibitory Smad7 protein (29). We further demonstrated that the transmembrane glycoprotein CD40, which has been shown to block or rescue B lymphocytes from TGF␤-induced apoptosis, can induce the expression of the TGF␤ signaling inhibitor Smad7. Thus, the pro-survival signal transduction pathway(s) activated by CD40 induce the expression of Smad7, which in turn, acts to down-regulate TGF␤ signaling (29).
In this study, we demonstrate that overexpression of the R-Smad, Smad3, sensitizes WEHI 231 B lymphocytes to the apoptotic effects of TGF␤. We show that TGF␤ specifically induces the expression of the pro-apoptotic protein Bim (Bcl-2interacting mediator of cell death), which is a BH3-only member of the Bcl-2 family. Bim induction by TGF␤ is accompanied by increased Bim/Bcl-2 heterodimerization and decreased mitochondrial membrane potential. Furthermore, we find that CD40 activation abrogates the TGF␤-mediated induction of Bim. These results suggest that the pro-apoptotic Bcl-2 family member Bim is a key mediator of the apoptotic response in WEHI 231 cells and that its expression is differentially regulated by either pro-or anti-apoptotic cytokines.

MATERIALS AND METHODS
Reagents-TGF␤2 was a generous gift from Genzyme Inc. (Cambridge, MA) and was used at a final concentration of 5 ng/ml. Purified hamster anti-mouse CD40 (␣-CD40), rabbit anti-Bim antibody, and mouse anti-Bad antibody were obtained from BD PharMingen (San Diego, CA). Goat anti-mouse IgM (␣-IgM) and mouse anti-Flag M2 antibodies, as well as reagent chemicals, were obtained from Sigma Chemical Co. Protease inhibitor mixture tablets and the DNA molecular weight standard (MWM XIV) were purchased from Roche Diagnostics (Indianapolis, IN). Mouse anti-Bcl-2 (C-2), mouse anti-Bax (B-9), rabbit anti-Bcl-X S/L (S-18), and rabbit anti-Hsp 90 (H-114) antibodies and normal rabbit IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-Smad3 antibody was from Zymed Labs (San Francisco, CA). Secondary antibodies were purchased from the following vendors: anti-mouse-IgG-HRP from Accurate Antibodies (San Diego, CA) and anti-rabbit-IgG-HRP from Bio-Rad. Oligonucleotide primers were obtained from Operon Technologies, Inc. (Alameda, CA). DiOC6 (3) was purchased from Molecular Probes, Inc. (Eugene, OR).
Cell Culture and Transfection-WEHI 231 cells were maintained in T75 flasks at a density of 2 ϫ 10 4 cells/ml in Dulbecco's modified Eagle's/F-12 medium supplemented with 5% fetal calf serum, 30 M 2-␤-mercaptoethanol, and antibiotics (100 units/ml of penicillin and 100 mg/ml of streptomycin). WEHI 231 clones that stably express FLAGtagged-Smad3 or FLAG-tagged-Smad3 dominant-negative (DN) proteins, as well as vector controls, were produced by retroviral infection, as previously described (29). The level of Smad3 or Smad3 DN expression was determined by immunoblotting cell lysates with anti-Smad3 or anti-FLAG M2 antibodies. Ba/F3 cells were maintained similar to WEHI 231 cells except that conditioned medium from WEHI 3B cells was added to a final concentration of 5% to provide the IL-3 essential for Ba/F3 survival.
Growth Inhibition Assay-WEHI 231 cells (1 ϫ 10 4 cells/ml) were cultured in T25 flasks at 37°C in the absence or presence of TGF␤ for up to 3 days. After each treatment, cells were collected, and viable cells that excluded trypan blue were counted.
Apoptosis Assays-Apoptosis was demonstrated by DNA ladder formation using either of the two following methods. Qualitative assess-ment of DNA ladder formation was performed by isolating oligonucleosomal DNA from cellular extracts and analyzing DNA by ethidium bromide staining after electrophoresis through 2.0% agarose gels, as described previously (29). Quantitative assessment of DNA ladder formation was performed using the Cell Death Detection ELISA plus kit (Roche Diagnostics, Indianapolis, IN). Briefly, WEHI 231 cells (20 ϫ 10 4 cells in 10 ml of medium) were cultured in T25 flasks at 37°C in the absence or presence of TGF␤ for up to 48 h. Cells were collected at the end of the experimental period and resuspended in 200 l of kit lysis buffer. The cellular lysate was centrifuged, and 20 l of the resulting supernatant was analyzed. Color development of the ELISA was monitored spectrophotometrically at 405 nm. Results are expressed as the ABS405 signal divided by the number of cells assayed. Apoptosis was also demonstrated by TUNEL, as described previously (29).
RNA Preparation and Northern Analysis-WEHI 231 cells (4 ϫ 10 6 cells in 40 ml of medium) were seeded into T75 flasks and treated for up to 8 h with TGF␤. The cells were collected by centrifugation, and RNA was isolated using an RNeasy kit from Qiagen (Valencia, CA). When 200 g of RNA was accumulated from several experiments, poly(A) ϩ RNA was isolated using an Oligotex mRNA Mini kit from Qiagen and used for Northern analysis. Northern analysis was carried out as described previously using 1% formaldehyde-agarose gels (29). The Bim and ␤-actin cDNA probes used in Northern analyses were obtained by RT-PCR using a Gene Amp PCR Core kit from PerkinElmer Life Sciences (Roche Applied Science). Briefly, 1 g of total RNA from Smad 3D WEHI 231 cells was reverse-transcribed using random primers. The cDNA template was denatured at 94°C, annealed at 48°C, and extended at 72°C for 1 min each and amplified for 30 cycles to obtain three Bim-specific PCR products of ϳ150, 250, and 450 bp. These three Bim PCR products likely arise by amplification of the three major Bim mRNA isoforms, BimEL, BimL, and BimS, as shown previously (34). The sequences of the Bim primers were 5Ј-TCTGAGTGTGACA-GAGAAGGTGGAC-3Ј for the forward primer and 5Ј-CAGCTCCTGTG-CAATCCGTATC-3Ј for the reverse primer. The cDNA template was denatured at 94°C, annealed at 60°C and amplified for 25 cycles to obtain a ␤-actin-specific 325-bp PCR product. The sequences of the ␤-actin primers were 5Ј-CCAAGGCCAACCGCGAGAAGATGAC-3Ј for the forward primer and 5Ј-AGGGTACATGGTGGTGCCGCCAGAC-3Ј for the reverse primer. The PCR products were purified using a Wizard PCR Prep column (Promega, Madison, WI) and 32 P-labeled using a Nick-translation kit (Roche Applied Science). Blots were hybridized overnight at 42°C in NorthernMax Hyb buffer (Ambion, Austin, TX) and washed 3 ϫ 20 min with 0.5ϫ SSC, 0.1% SDS at 55°C. Quantitation of 32 P-labeled probe hybridized to target mRNA transcripts on Northern blots was accomplished using a PhosphorImage analyzer (Molecular Dynamics, Sunnyvale, CA).
Nuclear Extract Preparation-Nuclear and cytosolic extract preparation for protein translocation experiments were performed as described previously (29). Typically, 2-5 ϫ 10 6 cells were resuspended 300 l of hypotonic buffer (10 mM HEPES, pH 8, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol plus protease inhibitors), allowed to swell on ice for 15 min, and lysed by the addition of 20 l of 10% Nonidet P-40 with vortexing. The extract was centrifuged at maximum speed for 1 min in a Beckman microfuge. The resulting supernatant was termed the cytoplasmic extract. The pellet was extracted in a high salt buffer (20 mM HEPES, pH 8, 25% glycerol, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol plus protease inhibitors) for 20 min followed by centrifugation for 10 min. The resulting supernatant was termed the nuclear extract. The protein concentration of the extracts was determined using Bradford's reagent (Pierce, Rockford, IL).
Mitochondrial Fractionation-Mitochondrial and cytosolic fractions were prepared from 1 ϫ 10 7 WEHI 231 cells using an ApoAlert cell fractionation kit from Clontech (Palo Alto, CA). Cells were treated in the absence or presence of TGF␤ for 24 h and collected by centrifugation. The cells were resuspended in 100 l of ice-cold fractionation buffer, incubated on ice for 30 min, and lysed by passing 50 times through a 26-gauge needle on a 0.5-ml syringe. The cytoplasmic and mitochondrial fractions were prepared from the lysates following the protocol supplied with the kit.
Western Blot Analysis-Western blot analysis was performed by standard SDS-PAGE, as described previously (29). Whole cell lysates were prepared from 2-5 ϫ 10 6 cells in 300 l of lysis buffer (20 mM Tris, pH 7.4, 1% Triton X-100, 10% glycerol, 137 mM NaCl, 2 mM EDTA, 1 mM Na 3 V0 4 , and protease inhibitors). Lysates were sonicated and clarified by centrifugation at 4°C for 10 min in a Beckman tabletop microcentrifuge at maximum speed. Typically, 25-50 g of whole cell lysates, 20 g of nuclear extracts, 50 g of mitochondrial fractions, or 50 -100 g of cytoplasmic extracts were separated on 10 or 12% acrylamide minigels and transferred to Immobilon-P membrane (Millipore, Bedford, MA). The membrane was blocked for 1 h in wash buffer (PBS containing 0.05% Tween 20) containing 5% nonfat dry milk followed by a 2-h incubation with primary antibody diluted in the same blocking buffer. After extensive washing, the blot was incubated with secondary antibody for 1 h in blocking buffer, washed, and processed using the ECL ϩ Western blotting detection system (Amersham Biosciences). Primary antibodies were employed at a 1:500 to 1:2000 dilution, and secondary antibodies were used at a 1:1000 to 1:5000 dilution.
Co-Immunoprecipitation-Whole cell lysates (500 g) were incubated overnight with 1 g of either rabbit anti-Bim antibody or normal rabbit IgG in 500 l of whole cell lysate extraction buffer containing protein G-agarose (Amersham Biosciences). The immune complexes were collected by centrifugation and washed extensively with whole cell lysate extraction buffer containing 500 mM NaCl. The presence of Bcl-2 in the immune complexes was determined by Western blotting.
Mitochondrial Depolarization Assay-Mitochondrial depolarization was measured by FACS using the lipophilic cation 3,3Ј-dihexyloxacarbocyanine iodide, DiOC6 (3) (35). Typically, 2 ϫ 10 6 cells were incubated for 24 h in the absence or presence of TGF␤, collected by centrifugation and resuspended in 300 l of phosphate-buffered saline containing 1% bovine serum albumin. DiOC6 (3) was added to a final concentration of 20 nM, and the cells were stained for 15 min at 37°C. Subsequently, the cells were placed on ice without washing and subjected to FACS analysis. FACS analyses were performed using a FACScan cytofluorometer (BD Biosciences). Cells were gated to eliminate forward and side scatter. DiOC6 (3) staining was monitored on 10,000 cells using the FL-1 channel.

Increased Smad3 Expression Sensitizes Cells to TGF␤-induced
Apoptosis-We have previously demonstrated that the inhibitory Smad7 protein blocks TGF␤-induced apoptosis in WEHI 231 B lymphocytes (29) suggesting a role for Smad proteins in mediating this signaling event. We sought therefore to more closely examine the role of the R-Smad proteins, Smad2 and Smad3, in mediating the apoptotic effects of TGF␤ in these cells. We retrovirally infected WEHI 231 cells to overexpress FLAG-tagged Smad2 and Smad3, and single cell-derived clones were selected in puromycin. While no Smad2overexpressing clones could be maintained, stable Smad3 overexpressing clones were obtained. Two Smad3-overexpressing clones, designated S3D and S3E, were chosen for further study. As shown in Fig. 1A, no endogenous Smad3 could be detected by Western blotting of cellular lysates (100 g of total protein) from vector control cells. The Smad3-overexpressing clones expressed different levels of Smad3, with S3D cells expressing higher levels than S3E cells.
We next chose to determine the phosphorylation status and subcellular localization of overexpressed Smad3 in the presence and absence of TGF␤. Fig. 1B demonstrates that both endogenous and exogenous Smad3 are maximally phosphorylated in response to TGF␤ within 30 -60 min and that phosphorylated Smad3 is present in the nucleus. The results also demonstrate that endogenous Smad3, although apparently expressed at very low levels in WEHI 231 cells, is readily detectable in nuclear fractions using the phosphospecific Smad3 antibody (Fig. 1B). We next chose to determine the subcellular localization of Smad3 in the overexpressing clones (S3D & S3E) under both basal and TGF␤-stimulated conditions. As shown in Fig. 1C, overexpressed Smad3 protein is present in both the cytosolic and nuclear fractions under resting conditions. In the presence of TGF␤, additional Smad3 translocates to the nucleus with a concomitant decrease in the cytoplasmic levels.
To determine the effect of Smad3 overexpression on TGF␤induced apoptosis, we first examined cellular viability following TGF␤ treatment. As shown in Fig. 2A, TGF␤ stimulation results in a decrease in cellular viability with time. S3D cells, which express the highest levels of Smad3 (Fig. 1A), show the greatest response to TGF␤ treatment, followed by the S3E and vector control transfectants. Also, as analyzed by ELISA (Fig.  2B) or by oligonucleosomal DNA ladder formation (Fig. 2C), there was a direct correlation between Smad3 expression levels and the extent of TGF␤-induced apoptosis. S3D cells, which express the highest levels of exogenous Smad3 (Fig. 1A), showed the greatest sensitivity to the apoptotic effects of TGF␤.
TGF␤ Induces Bim Protein Expression-Previous reports have implicated members of the Bcl-2 family of proteins in mediating the apoptotic effects of TGF␤ (27,33). We therefore examined whether TGF␤-mediated apoptosis in the Smad3overexpressing clones might be associated with changes in the expression profile of Bcl-2 family members. As shown in Fig.  3A, immunoblot analysis of cellular lysates from Smad3-overexpressing S3D cells revealed that TGF␤ treatment elicited its most significant effect on the pro-apoptotic family member Bim. While inhibitory effects where observed on the expression levels of Bad, Bax, and Bcl-2 following a 24-h TGF␤ treatment, these could be secondary to the apoptotic state of the cells. Bim expression levels, however, were significantly increased as early as 2 h after TGF␤ addition and typically continued to increase for at least 24 h in the presence of TGF␤ (Fig. 3A). Lysates were also tested for the presence of Bcl-X S/L by immunoblot analysis, but no bands were detectable. The levels of a cytosolic heat shock protein, Hsp-90, were also analyzed and served as a protein-loading control.
The results shown in Fig. 3B demonstrate that while TGF␤ induces Bim protein expression in the parental WEHI-231 cells, the magnitude and kinetics of the induction were not the same as in the Smad3-overexpressing S3D and S3E clones. TGF␤-mediated Bim induction in the S3D clone was on the order of 16-fold following a 48-h TGF␤ treatment, while in the parental WEHI-231 cells Bim induction was ϳ3-4-fold above non-stimulated levels (Fig. 3B). Also, whereas TGF␤-mediated induction of Bim protein was reliably observed within 4 h in the S3D clone (Fig. 3A), its induction in parental WEHI 231 cells was delayed and not observed until 24 h after TGF␤ addition (data not shown). These results are consistent with the reduced TGF␤-induced apoptosis observed in parental versus Smad3 overexpressing cells, as shown in Fig. 2. The results of Fig. 3B also demonstrate that induction of Bim expression by TGF␤ occurs in two independent Smad3-overexpressing clones (S3D and S3E), thus suggesting that the observed TGF␤-mediated induction of Bim in the S3D clone is not due to clonal selection. These results demonstrate that Smad3 overexpression re-sults in a more rapid and robust induction of Bim protein, which may be responsible for potentiating the apoptotic effects of TGF␤.
Previous studies have demonstrated increased Bim expression in the mouse pre-B cell line, Ba/F3, during apoptosis induced by IL-3 withdrawal (36). We therefore wished to examine whether TGF␤ also induced Bim expression and apoptosis in Ba/F3 cells. As shown in Fig. 3, TGF␤ treatment of Ba/F3 cells resulted in a time-dependent increase in oligonucleosomal DNA ladder formation (Fig. 3C) and Bim protein expression (Fig. 3D). Following an 8 h TGF␤ treatment there was a 4.5-fold induction in Bim protein levels above control, non-stimulated levels (Fig. 3D). Thus, in two progenitor Blymphocyte cell lines, WEHI-231 and Ba/F3, TGF␤ induces Bim protein expression, providing further support for the crucial role of Bim in TGF␤-induced apoptosis in B lymphocytes.
Further support for the role of Smad3 in mediating Bim Vector control, Smad3D, and Smad3E cells were incubated in the absence (Ϫ) or presence (ϩ) of TGF␤ for 24 h. A small aliquot of each sample was saved for cell counts and the majority of the cells was used to quantitate apoptosis by ELISA. Results are expressed as the absorbance reading obtained from the ELISA normalized to the cell count. C, TGF␤-induced DNA ladder formation. Vector control or Smad3D cells were incubated in the absence (Ϫ) or presence (ϩ) of TGF␤ overnight. Oligonucleosomal DNA was isolated, electrophoresed through a 2% agarose gel, and stained with ethidium bromide. A 100-bp DNA ladder standard was run with the samples and is shown at the left. induction by TGF␤ is provided by the results of Fig. 4A demonstrating the effects of overexpression of dominant-negative forms of Smad3 on TGF␤ induction of Bim. In two distinct clones, S3.3DN and S3.8DN, TGF␤-induced Bim protein expression is dramatically reduced (lower panels) compared with the level of Bim induction by TGF␤ in the Smad3-overexpressing clones, S3D and S3E (upper panels). The expression level of dominant-negative Smad3 in the S3.3DN and S3.8DN clones is shown in Fig. 4, panel B. The results shown in Fig. 4C show that TGF␤-mediated apoptosis in these two clones, overexpressing dominant-negative Smad3, is reduced relative to parental WEHI 231 cells, in agreement with our previous study (29). Taken together, these results suggest that the induction of Bim expression correlates with the TGF␤ apoptotic response in WEHI 231 cells and is potentiated by overexpression of Smad3.
TGF␤ Induces Bim mRNA Expression-To determine whether the up-regulation of Bim expression by TGF␤ was a result of enhanced transcription, Bim mRNA was analyzed (Fig. 5A). Bim has been shown to have multiple isoforms generated by alternative splicing (34,37,38) with three predominant isoforms, termed BimS, BimL, and BimEL for Bim short (S), long (L), and extra long (EL). As depicted in Fig. 5A, we detected several Bim transcripts, in agreement with previous reports (34), and expression of several of these mRNAs is significantly elevated by TGF␤ addition. In particular, the largest transcript (Fig. 5A, upper arrow) is induced greater than 3-fold following a 4 -8-h TGF␤ treatment (Fig. 5B). The lower, most prominent transcript (Fig. 5A, bottom arrow), did not show any significant induction by TGF␤. While Fig. 5A demonstrates that TGF␤ treatment induced the expression of several other less prominent transcripts, these transcripts were not reliably observed by Northern blot analysis. In order to determine whether TGF␤-induced Bim protein expression required new protein synthesis or mRNA transcription, S3D cells were co-incubated with cycloheximide or actinomycin D, respectively, during TGF␤ treatment. As shown in Fig. 5C, both cycloheximide and actinomycin D treatment inhibited Bim protein expression induced by TGF␤. These two inhibitors had no effect on either Bcl-2 or Hsp-90 protein levels, demonstrating that the inhibition of Bim expression was not due to a nonspecific or toxic effect. Taken together, the results of Fig. 5 are consistent with the idea that TGF␤-induced Bim expression occurs through a transcriptional mechanism.
TGF␤ Promotes Mitochondrial Bim Accumulation, Bim Heterodimerization with Bcl-2, and Loss of Mitochondrial Membrane Potential-Bim has previously been reported to translocate to the mitochondria and form heterodimers with other Bcl-2 family members in response to apoptotic stimuli (34,39). It was of interest, therefore, to determine the subcellular location of Bim induced by TGF␤ treatment. We performed immunoblot analysis on subcellular fractions of S3D cells treated in the absence or presence of TGF␤ for 24 h. As shown in Fig. 6A, TGF␤ induces the expression of Bim protein and has relatively little effect on Bcl-2 expression levels in whole cell lysates (WCL). In mitochondrial fractions, there is a significant increase in Bim protein levels in the presence of TGF␤ and again little change in Bcl-2 levels in the absence or presence of TGF␤. The results also demonstrate that there is no significant amount of either Bim or Bcl-2 in the cytoplasm and that the two fractions are not cross-contaminated, as indicated by the lack of the cytosolic protein Hsp-90 in the mitochondrial fraction and the absence of the mitochondrial protein Bcl-2 in the cytosolic fraction.
Since TGF␤ promotes accumulation of Bim in the mitochondria, we wished to determine whether TGF␤ could also promote heterodimerization of Bim with Bcl-2. The results of Fig. 6B demonstrate by co-immunoprecipitation analysis that TGF␤ treatment induces, in a time-dependent manner, complex formation between Bim and Bcl-2. There is relatively little Bim associated with Bcl-2 under basal conditions but TGF␤ stimulation promotes the association of Bim with Bcl-2, with maximal effects observed between 8 and 24 h. Western blot analysis of the lysates used for immunoprecipitation revealed a gradual, time-dependent reduction in Bcl-2 and Hsp-90 protein levels.
Bim protein has previously been shown to disrupt mitochondrial membrane potential and promote the release of mitochondrial cytochrome c, both early and critical events in many apoptotic processes (40). It was of interest, therefore, to determine whether TGF␤ stimulation alters mitochondrial membrane potential in S3D cells. We used the lipophilic cationic dye DiOC6 (3) Fig. 6C demonstrate that TGF␤ induces a decrease of DiOC6 staining following a 24-h treatment compared with untreated, control cells. This depolarization is more apparent when the two histograms are overlaid, shown to the right of Fig. 6C. The M2 region of the histograms represents healthy, propidium iodide-negative cells, whereas the M1 region represents damaged, propidium iodide-positive cells. Taken together, these results indicate that TGF␤ promotes an increase in mitochondrial Bim protein levels, resulting in an increased heterodimerization with Bcl-2 and a concomitant loss of mitochondrial membrane potential.

CD40 Stimulation Antagonizes TGF␤-mediated Apoptosis, Bim Protein Expression, and Heterodimerization with Bcl-2-
The transmembrane glycoprotein CD40 has been shown to couple to multiple signaling pathways and its activation plays a critical role in promoting cellular survival in numerous cell types, including WEHI 231 B lymphocytes (29). In Fig. 7 we demonstrate by ELISA (Fig. 7A) and by oligonucleosomal DNA ladder formation (Fig. 7B) that activation of CD40 by ␣-CD40 antibody is able to rescue or abrogate the apoptotic effects of TGF␤ in S3D cells. We next determined whether CD40 rescue of TGF␤-mediated apoptosis was also associated with effects on Bim protein expression. The immunoblot analysis shown in Fig. 7C demonstrates that co-stimulation of cells with ␣-CD40 and TGF␤ inhibits the induction of Bim protein mediated by TGF␤. The induction of Bim protein following a 6-and 24-h TGF␤ treatment is inhibited to near basal levels following co-stimulation with ␣-CD40. Furthermore, the results of Fig.  7D demonstrate that the TGF␤-induced complex formation between Bim and Bcl-2 following a 6-and 24-h TGF␤ treatment is inhibited by ␣-CD40. These data demonstrate that CD40 stimulation can rescue Smad3-overexpressing WEHI 231 cells from TGF␤-mediated apoptosis and suggest that inhibition of Bim protein induction by CD40 stimulation may represent a mechanism by which pro-survival cytokines suppress the TGF␤ apoptotic response.

DISCUSSION
Hematopoiesis is governed by a balance between opposing cell death and survival programs that are, in turn, regulated by survival factors and cytokines. It is well established that the Bcl-2 family, both pro-and anti-apoptotic members, plays a crucial role in regulating these programs (41,42). Bim, the Bcl-2 interacting mediator of cell death, is a recently discovered BH3-only member of the Bcl-2 family which is expressed in hematopoietic tissues, as well as in epithelial, neuronal, and germ cells (34,43). Similar to other BH3-only family members, Bim is thought to induce cell death by binding to and neutralizing pro-survival Bcl-2 family members, thereby releasing Bax-like proteins to execute cell death (44,45). A specific role for Bim in mediating hematopoietic cell death was demonstrated in lymphocytes isolated from Bim knockout mice. Both B and T lymphocytes from Bim(Ϫ/Ϫ) mice survive 10 -30 times better than wild-type cells following cytokine withdrawal, as well as after several other apoptotic stimuli (46,47). The cell line used in our study, WEHI 231 cells, is an immature B-cell line that is used extensively as a model of B cell tolerance and apoptosis. Thus, these cells represent an attractive in vitro model system to study the role of Bim in immunoregulation.
Here we show that TGF␤ induces the expression of Bim in FIG. 5. TGF␤ induces Bim mRNA expression. A, Northern analysis of Bim mRNA levels. Smad3D cells were treated in the absence (Cont.) or presence of TGF␤ for up to 8 h. Poly(A) ϩ RNA was isolated, and Bim mRNA transcripts were detected by Northern analysis, as described under "Materials and Methods." The blot was then stripped and analyzed for ␤-actin mRNA transcript levels by Northern analysis. The arrows to the right of the Bim blot indicate the Bim mRNA transcripts that were used for quantitation. B, quantitation of Bim mRNA transcript levels. The amount of radioactivity in the two Bim transcripts and in the ␤-actin transcript of panel A was quantitated by phosphorimage analysis. The amount of radioactivity in the upper and lower Bim mRNA transcripts was divided by the amount of radioactivity in the corresponding ␤-actin transcript. The ratio obtained for the untreated control (Cont.) sample was set at 100%, and the ratios obtained for the TGF␤-treated samples were normalized to this control value. The results for the upper and lower Bim transcript levels are represented by white and black bars, respectively. C, effect of protein and mRNA synthesis inhibitors on TGF␤-induced Bim protein expression. Smad3D cells were treated in the absence (Ϫ) or presence (ϩ) of TGF␤ for 8 h. During the last 4 h of TGF␤ treatment, either cycloheximide (10 g/ml) or actinomycin D (0.5 g/ ml) was also present. Whole cell lysates were prepared, and 50-g aliquots analyzed for the presence of Bim, Bcl-2, and Hsp-90 by Western blotting.

WEHI 231 cells and that this induction is potentiated in Smad3-overexpressing cells.
Previous studies of BH3-only proteins have demonstrated that their apoptotic function may be regulated by several different mechanisms (48). The apoptotic function of Bad is regulated through phosphorylation of two specific serine residues that abrogate its binding to and neutralizing of the pro-survival proteins Bcl-2 or Bcl-X L (49 -51). Bid, however, is proteolytically cleaved by active caspase 8, generating an active product termed tBid, for truncated Bid, that translocates to the mitochondria and induces apoptosis (52). Control of subcellular localization has also been proposed as a regulatory mechanism for BH3-only proteins, including Bim (39). Bim is sequestered to the microtubular motor complex by its binding to dynein light chain (LC8) and following pro-apoptotic stimuli is released into the cytoplasm allowing its interaction with prosurvival Bcl-2 family members (39). More recent reports, demonstrate that cytokine modulation of Bim expression levels represent another mechanism of regulating the apoptotic function of BH3-only proteins (36,53). Specifically, IL-3 withdrawal from murine hematopoietic progenitor cells results in an upregulation of Bim expression with an associated induction of apoptosis (36,53). Similar results are obtained when NGF is withdrawn from cultured neuronal cells (45,54,55).
Our results presented here corroborate a model in which Bim expression levels mediate cytokine regulated cell death. However, as opposed to negative regulation of Bim expression levels by the pro-survival cytokines IL-3 or NGF, we demonstrate that addition of a pro-apoptotic cytokine, TGF␤, results in the up-regulation of Bim expression levels in two different B-cell lines, WEHI 231 and Ba/F3. This is the first demonstration that addition, and not withdrawal, of a cytokine results in enhanced Bim expression. We further demonstrate that the pro-survival cytokine CD40 is capable of inhibiting the induction of Bim expression in WEHI 231 cells in response to TGF␤ concomitant with its rescue of the cells from TGF␤-mediated apoptosis. Thus both pro-and anti-apoptotic cytokines regulate Bim expression levels in WEHI 231 cells and underscore the pivotal role of this molecule in cytokine regulation of cell survival and apoptosis. Previous studies have implicated several signaling pathways as mediating IL-3-induced repression of Bim expression levels in hematopoietic cells, specifically Ba/F3 cells. Two Ras-activated pathways, involving Raf/MAPK and PI3K/mTOR, were shown to be transducers of an IL-3-dependent down-regulation of Bim expression concomitant with cell survival (53). Several reports have also demonstrated that IL-2 and IL-3 regulate phosphorylation of the forkhead family (FKHR) of transcriptional regulators in a PI3K/AKT/PKB-dependent fashion to promote cell survival through repression of Bim expression FIG. 6. TGF␤ promotes mitochondrial Bim accumulation, heterodimerization with Bcl-2, and loss of mitochondrial membrane potential. A, subcellular localization of Bim. Smad3D cells were treated in the absence (Ϫ) or presence (ϩ) of TGF␤ for 24 h. A small portion of the cells from each sample was used to make a whole cell lysate whereas the majority of the cell sample was used to prepare a mitochondrial and cytoplasmic fraction. Aliquots of whole cell lysate (WCL, 50 g), mitochondrial fraction (Mito., 50 g) and cytosolic fraction (Cyto., 100 g) were analyzed by Western blotting for the presence of Bim, Bcl-2, and Hsp-90. B, co-immunoprecipitation of Bim and Bcl-2. Smad3D cells were treated in the absence (Cont.) or presence of TGF␤ for up to 24 h, and whole cell lysates prepared. Bim protein was immunoprecipitated from 500 g of whole cell lysate and immunoprecipitates were analyzed for the presence of Bcl-2 by Western blotting (upper blot). Some lysates were immunoprecipitated with normal rabbit IgG as a negative control. In addition, the whole cell lysates (50 g) were directly analyzed by Western blotting for the presence of Bcl-2 (middle blot) and Hsp-90 (lower blot). C, mitochondrial depolarization induced by TGF␤. Smad3D cells were incubated in the absence (Control) and presence of TGF␤ for 24 h. The cells were collected and stained with DiOC6, as described under "Materials and Methods." The plot on the right is an overlay of the two individual plots shown on the left of fluorescence versus cell count for the control and TGF␤ samples. levels (36,56). IL-3 was shown to negatively regulate FKHR-L1 through phosphorylation of Thr-32 and Ser-253 on FKHR-L1 correlating with a down-regulation of Bim expression (36). Furthermore, inducible expression of exogenous FKHR-L1 resulted in an elevation of Bim expression levels and induction of apoptosis, suggesting that Bim expression is directly regulated by FKHR-L1 (36,57).
In this study, we demonstrate that TGF␤-mediated induction of Bim expression is potentiated in Smad3-overexpressing WEHI 231 cells. While it is well established that Smad proteins are key signaling components in TGF␤-mediated apoptosis, their precise role in regulating this cellular process is still unclear (29, 33, 58 -61). We have previously shown that Smad7, the inhibitory Smad protein, abrogates TGF␤-induced apoptosis in WEHI 231 cells (29). Studies have also shown that overexpression of wild-type Smad3 induces apoptosis in human lung epithelial cells (58) and that overexpression of dominantnegative forms of Smad3 inhibit TGF␤ induced apoptotic cell death in Hep3B cells (63). More recently, TGF␤-induced cell death in rat FAO cells was shown to be potently enhanced by overexpression of Smad3 and blocked by antisense Smad3 RNA expression (33). In this same report, it was shown that TGF␤ induced the cleavage of the BH3-only protein BAD through an as yet to be determined Smad3-dependent mechanism (33).
The findings presented here are noteworthy in that they identify the pro-apoptotic protein Bim as a potential transcriptional target for TGF␤ that is regulated in a Smad-dependent manner. Overexpression of Smad3 results in a sensitization of WEHI 231 B lymphocytes to TGF␤-mediated apoptosis (Fig. 1) concomitant with the induction of Bim mRNA (Fig. 5) and enhanced expression of Bim protein (Figs. 3, 4, and 6). Dominant-negative interfering forms of Smad3 block TGF␤-mediated cell death (29) and induction of Bim protein expression (Fig. 4). The molecular mechanism through which Smad3 may mediate Bim expression requires further investigation. It is of note that members of the FKHR family of transcription factors were the first Smad-interacting factors isolated and have subsequently been demonstrated to serve as Smad transcriptional co-factors regulating, in both a positive and negative manner, TGF␤ transcriptional responses (64 -66). Computer analysis of the 5Ј regulatory region of the murine Bim gene (67) identified many putative Smad-DNA binding elements (SBE), as well as consensus FKHR binding sequences. These data suggest that combinatorial interactions between Smad3 and FKHR family members could play a regulatory role in transcriptional activation of the Bim promoter.
Our results also demonstrate that the pro-survival pathway induced by CD40 results in abrogation of TGF␤-mediated apoptosis and Bim expression (Fig. 7). Interestingly, CD40 has previously been shown to mediate its pro-survival effects in B lymphocytes through the PI3K/AKT pathway (29,68,62), similar to that described for the IL-3 system. It is not clear however, whether downstream of PI3K/AKT, CD40 regulates FKHR transcriptional activation, as has been demonstrated for IL-3. Thus, it appears that repression of Bim expression may be one of the central mechanisms by which cytokines signaling through the PI3K/AKT pathway mediate cell survival. It will be of interest to investigate the potential role of forkhead tran-FIG. 7. CD40 antagonizes TGF␤-mediated events. A, CD40 rescue of TGF␤-induced apoptosis. Smad3D cells were untreated (Cont.) or treated for 24 h with TGF␤ alone, CD40 alone or both TGF␤ and CD40. A small aliquot of each sample was saved for cell counts, and the majority of the cells was used to quantitate apoptosis by ELISA. Results are expressed as the absorbance reading obtained from the ELISA normalized to the cell count. B, CD40 rescue of TGF␤-induced DNA ladder formation. Smad3D cells were untreated (Cont.) or treated overnight with TGF␤ alone, CD40 alone, or both TGF␤ and CD40. Oligonucleosomal DNA was isolated, electrophoresed through a 2% agarose gel, and stained with ethidium bromide. A 100-bp DNA ladder standard was run with the samples and is shown at the left. C, CD40 blocks TGF␤-induced Bim protein expression. Smad3D cells were untreated (Cont.) or treated for 6 or 24 h with TGF␤ alone, CD40 alone, or both TGF␤ and CD40. Whole cell lysates were prepared and 50-g aliquots were analyzed by Western blotting for the presence of Bim (upper blot) or Hsp-90 (lower blot). D, CD40 blocks TGF␤-induced Bim/Bcl-2 binding. Lysates (500 g) used in panel C, were immunoprecipitated with anti-Bim antibody or normal rabbit IgG. The immunoprecipitates were analyzed by Western blotting for the presence of Bcl-2. scription factors as a convergence point between the TGF␤/ Smad3-mediated cell death pathway and the CD40-mediated survival pathway in future studies.