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

Gary M. Wildey, Supriya Patil, and Philip H. HoweDagger

From the Department of Cell Biology, The Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195

Received for publication, November 22, 2002, and in revised form, March 6, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Transforming growth factor-beta (TGFbeta ) 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 TGFbeta -induced apoptosis and expression of the pro-apoptotic protein Bim in WEHI 231 B lymphocytes. Overexpression of dominant-negative forms of Smad3 abrogate these TGFbeta -induced responses. We also demonstrate that TGFbeta 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 TGFbeta 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 TGFbeta -mediated Bim induction. Our data provide the first evidence of Bim expression levels that are increased by the addition of a pro-apoptotic cytokine, TGFbeta , and also suggest that the TGFbeta -specific transcription factor Smad3 plays a role in mediating Bim expression levels and apoptosis.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Transforming growth factor beta  (TGFbeta )1 and its related factors modulate essential cellular functions ranging from cellular proliferation and differentiation to apoptosis (1-4). Signaling by TGFbeta 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 (Tbeta RII) promotes complex formation with the type I receptor (Tbeta RI/ALK5). Subsequent phosphorylation and activation of Tbeta RI/ALK5 by Tbeta RII leads to further propagation of TGFbeta signaling by several signaling cascades, which include the Smads, MAPK, and PI3K (1-4).

Signaling by TGFbeta 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 (1-4). 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 TGFbeta receptors. Recently, we have shown that the adaptor molecule disabled-2 (Dab2) links TGFbeta 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, TGFbeta has also been reported to signal through components of the MAPK and PI3K/Akt pathways. TGFbeta has been shown to activate extracellular signal-regulated kinase (ERK) (9, 10), Jun N-terminal kinase (JNK) (11-14), p38 mitogen-activated protein kinase (p38) (15, 16), and PI3K/AKT (17). The TGFbeta 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 TGFbeta receptors to these signaling pathways (11, 18-20) or that activation of these pathways may be indirect, possibly resulting from Smad-dependent transcriptional responses.

TGFbeta 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 (23-25), hematopoietic cells (26), and in B lymphocytes (27-29). The molecular mechanisms mediating the pro-apoptotic effects of TGFbeta 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 Tbeta RII and couples TGFbeta signaling to the apoptotic machinery in AML12 hepatocytes (30). In the Hep3B hepatoma cell line, TGFbeta 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 TGFbeta signaling pathway), a septin-like protein, translocates from the mitochondria to the nucleus in response to TGFbeta treatment of the prostatic epithelial cell line NRP-154 (32). The Bcl-2 family of proteins has also been implicated as mediators of TGFbeta -induced apoptosis. Early studies in the WEHI 231 B lymphocyte cell line demonstrated that stable overexpression of Bcl-XL abrogated TGFbeta -mediated apoptosis (27). More recently, it was shown in the FaO rat hepatoma cell line that TGFbeta 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 TGFbeta -mediated apoptosis and BAD cleavage (33).

We have recently shown that TGFbeta -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 TGFbeta -induced apoptosis, can induce the expression of the TGFbeta 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 TGFbeta signaling (29).

In this study, we demonstrate that overexpression of the R-Smad, Smad3, sensitizes WEHI 231 B lymphocytes to the apoptotic effects of TGFbeta . We show that TGFbeta specifically induces the expression of the pro-apoptotic protein Bim (Bcl-2-interacting mediator of cell death), which is a BH3-only member of the Bcl-2 family. Bim induction by TGFbeta is accompanied by increased Bim/Bcl-2 heterodimerization and decreased mitochondrial membrane potential. Furthermore, we find that CD40 activation abrogates the TGFbeta -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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents-- TGFbeta 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 (alpha -CD40), rabbit anti-Bim antibody, and mouse anti-Bad antibody were obtained from BD PharMingen (San Diego, CA). Goat anti-mouse IgM (alpha -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-XS/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 × 104 cells/ml in Dulbecco's modified Eagle's/F-12 medium supplemented with 5% fetal calf serum, 30 µM 2-beta -mercaptoethanol, and antibiotics (100 units/ml of penicillin and 100 mg/ml of streptomycin). WEHI 231 clones that stably express FLAG-tagged-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 × 104 cells/ml) were cultured in T25 flasks at 37 °C in the absence or presence of TGFbeta 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 assessment 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 ELISAplus kit (Roche Diagnostics, Indianapolis, IN). Briefly, WEHI 231 cells (20 × 104 cells in 10 ml of medium) were cultured in T25 flasks at 37 °C in the absence or presence of TGFbeta for up to 48 h. Cells were collected at the end of the experimental period and resuspended in 200 µl of kit lysis buffer. The cellular lysate was centrifuged, and 20 µl 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 × 106 cells in 40 ml of medium) were seeded into T75 flasks and treated for up to 8 h with TGFbeta . The cells were collected by centrifugation, and RNA was isolated using an RNeasy kit from Qiagen (Valencia, CA). When 200 µg of RNA was accumulated from several experiments, poly(A)+ 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 beta -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 µg 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 ~150, 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 5'-TCTGAGTGTGACAGAGAAGGTGGAC-3' for the forward primer and 5'-CAGCTCCTGTGCAATCCGTATC-3' for the reverse primer. The cDNA template was denatured at 94 °C, annealed at 60 °C and amplified for 25 cycles to obtain a beta -actin-specific 325-bp PCR product. The sequences of the beta -actin primers were 5'-CCAAGGCCAACCGCGAGAAGATGAC-3' for the forward primer and 5'-AGGGTACATGGTGGTGCCGCCAGAC-3' for the reverse primer. The PCR products were purified using a Wizard PCR Prep column (Promega, Madison, WI) and 32P-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 × 20 min with 0.5× SSC, 0.1% SDS at 55 °C. Quantitation of 32P-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 × 106 cells were resuspended 300 µl 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 µl 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 × 107 WEHI 231 cells using an ApoAlert cell fractionation kit from Clontech (Palo Alto, CA). Cells were treated in the absence or presence of TGFbeta for 24 h and collected by centrifugation. The cells were resuspended in 100 µl 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 × 106 cells in 300 µl of lysis buffer (20 mM Tris, pH 7.4, 1% Triton X-100, 10% glycerol, 137 mM NaCl, 2 mM EDTA, 1 mM Na3V04, 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 µg of whole cell lysates, 20 µg of nuclear extracts, 50 µg of mitochondrial fractions, or 50-100 µg 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+ 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 µg) were incubated overnight with 1 µg of either rabbit anti-Bim antibody or normal rabbit IgG in 500 µl 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 3,3'-dihexyloxacarbocyanine iodide, DiOC6 (3) (35). Typically, 2 × 106 cells were incubated for 24 h in the absence or presence of TGFbeta , collected by centrifugation and resuspended in 300 µl 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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Increased Smad3 Expression Sensitizes Cells to TGFbeta -induced Apoptosis-- We have previously demonstrated that the inhibitory Smad7 protein blocks TGFbeta -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 TGFbeta 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 Smad2-overexpressing 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 µg 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.


View larger version (49K):
[in this window]
[in a new window]
 
Fig. 1.   Overexpressed Smad3 levels in WEHI 231 B lymphocytes. A, basal Smad3 protein levels. Whole cell lysates were prepared from vector control cells (Cont.) and from two Smad3-overexpressing clones, termed Smad3D (S3D) and Smad3E (S3E). The level of Smad3 protein (S3) in 10-100 µg of whole cell lysates was determined by Western blotting using an anti-Smad3 polyclonal antibody. B, TGFbeta -induced Smad3 phosphorylation. Vector control cells and Smad3D cells were incubated in the absence (0) or presence of TGFbeta for the indicated times. At the end of the incubation nuclear fractions were prepared and the level of phosphorylated Smad3 protein (S3P) in 50 µg of nuclear fractions was determined by Western blotting using a phosphospecific anti-Smad3 polyclonal antibody. C, TGFbeta -induced nuclear translocation of Smad3. Vector control, Smad3D, and Smad3E cells were incubated in the absence (-) or presence (+) of TGFbeta for 1 h. Nuclear and cytoplasmic fractions were prepared, and the level of Smad3 protein (S3) was determined by Western blotting using an anti-Smad3 polyclonal antibody.

We next chose to determine the phosphorylation status and subcellular localization of overexpressed Smad3 in the presence and absence of TGFbeta . Fig. 1B demonstrates that both endogenous and exogenous Smad3 are maximally phosphorylated in response to TGFbeta 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 TGFbeta -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 TGFbeta , additional Smad3 translocates to the nucleus with a concomitant decrease in the cytoplasmic levels.

To determine the effect of Smad3 overexpression on TGFbeta -induced apoptosis, we first examined cellular viability following TGFbeta treatment. As shown in Fig. 2A, TGFbeta 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 TGFbeta 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 TGFbeta -induced apoptosis. S3D cells, which express the highest levels of exogenous Smad3 (Fig. 1A), showed the greatest sensitivity to the apoptotic effects of TGFbeta .


View larger version (41K):
[in this window]
[in a new window]
 
Fig. 2.   Increased Smad3 expression sensitizes cells to TGFbeta -induced growth arrest and apoptosis. A, TGFbeta -induced growth inhibition. Vector control (Cont.), Smad3D (S3D), and Smad3E (S3E) cells were seeded into T75 flasks (Day 0) and incubated in the absence (-) or presence (+) of TGFbeta for up to 3 days without any change of the tissue culture medium. 1, 2, or 3 days after seeding, the cells were collected and viable cell counts determined. B, TGFbeta -induced apoptosis. Vector control, Smad3D, and Smad3E cells were incubated in the absence (-) or presence (+) of TGFbeta 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, TGFbeta -induced DNA ladder formation. Vector control or Smad3D cells were incubated in the absence (-) or presence (+) of TGFbeta 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.

TGFbeta Induces Bim Protein Expression-- Previous reports have implicated members of the Bcl-2 family of proteins in mediating the apoptotic effects of TGFbeta (27, 33). We therefore examined whether TGFbeta -mediated apoptosis in the Smad3-overexpressing 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 TGFbeta 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 TGFbeta 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 TGFbeta addition and typically continued to increase for at least 24 h in the presence of TGFbeta (Fig. 3A). Lysates were also tested for the presence of Bcl-XS/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.


View larger version (59K):
[in this window]
[in a new window]
 
Fig. 3.   TGFbeta induces Bim protein expression. A, TGFbeta -induced changes in the protein level of Bcl-2 family members. Smad3D cells were incubated in the absence (Cont.) or presence of TGFbeta for up to 24 h. Whole cell lysates were prepared and 50-µg aliquots were analyzed for the presence of Bim, Bad, Bax, Bcl-2, and Hsp-90 by Western blotting. B, quantitation of TGFbeta -induced increases in Bim protein levels. Parental control (Cont.), Smad3D (S3D), and Smad3E (S3E) cells were treated in the absence or presence of TGFbeta for 24 or 48 h. Whole cell lysates were prepared and analyzed for the presence of Bim by Western blotting. The high molecular weight isoform of Bim detected by Western blotting was quantitated using Adobe Photoshop. Results show the fold-increase over untreated controls. C, TGFbeta induces apoptosis in Ba/F3 cells. Wild-type Ba/F3 cells were incubated in the absence (Cont.) or presence of TGFbeta for up to 24 h. At the times indicated oligonucleosomal DNA was isolated, electrophoresed through 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. D, TGFbeta induces Bim protein expression in Ba/F3 cells. Ba/F3 cells were treated in the absence (Cont.) or presence of TGFbeta for the times indicated, and 50 µg of whole cell lysates were analyzed for the expression of Bim and Hsp-90 by Western blotting.

The results shown in Fig. 3B demonstrate that while TGFbeta 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. TGFbeta -mediated Bim induction in the S3D clone was on the order of 16-fold following a 48-h TGFbeta treatment, while in the parental WEHI-231 cells Bim induction was ~3-4-fold above non-stimulated levels (Fig. 3B). Also, whereas TGFbeta -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 TGFbeta addition (data not shown). These results are consistent with the reduced TGFbeta -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 TGFbeta occurs in two independent Smad3-overexpressing clones (S3D and S3E), thus suggesting that the observed TGFbeta -mediated induction of Bim in the S3D clone is not due to clonal selection. These results demonstrate that Smad3 overexpression results in a more rapid and robust induction of Bim protein, which may be responsible for potentiating the apoptotic effects of TGFbeta .

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 TGFbeta also induced Bim expression and apoptosis in Ba/F3 cells. As shown in Fig. 3, TGFbeta 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 TGFbeta treatment there was a 4.5-fold induction in Bim protein levels above control, non-stimulated levels (Fig. 3D). Thus, in two progenitor B-lymphocyte cell lines, WEHI-231 and Ba/F3, TGFbeta induces Bim protein expression, providing further support for the crucial role of Bim in TGFbeta -induced apoptosis in B lymphocytes.

Further support for the role of Smad3 in mediating Bim induction by TGFbeta is provided by the results of Fig. 4A demonstrating the effects of overexpression of dominant-negative forms of Smad3 on TGFbeta induction of Bim. In two distinct clones, S3.3DN and S3.8DN, TGFbeta -induced Bim protein expression is dramatically reduced (lower panels) compared with the level of Bim induction by TGFbeta 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 TGFbeta -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 TGFbeta apoptotic response in WEHI 231 cells and is potentiated by overexpression of Smad3.


View larger version (38K):
[in this window]
[in a new window]
 
Fig. 4.   Smad3 dominant-negative abrogates TGFbeta -induced Bim protein expression. A, effect of TGFbeta on Bim protein levels in WEHI 231 clones overexpressing Smad3 dominant-negative protein. Smad3D (S3D) and Smad3E (S3E) cells as well as two independent WEHI 231 clones that overexpressed dominant-negative Smad3 (S3.3DN and S3.8DN) were treated in the absence (Cont.) or presence of TGFbeta for 24 or 48 h. Whole cell lysates were prepared, and 50 µg were analyzed by Western blotting for either Bim or Hsp-90. B, quantitation of Smad3 dominant-negative protein levels. Whole cell lysates were prepared from vector control (Cont.) cells and the two Smad3 dominant-negative overexpressing clones (S3.8DN and S3.3DN), as well as from COS cells transiently transfected with dominant-negative Smad3 and analyzed by Western blotting for the presence of exogenous dominant-negative Smad3 using an anti-Smad 3 antibody. C, TGFbeta -induced apoptosis in Smad3 dominant-negative clones. Vector control cells (Cont.) as well as S3.3DN and S3.8DN cells were treated in the absence or presence of TGFbeta for 24 h. Apoptosis was determined by the percentage of TUNEL (+) cells, as described under "Materials and Methods."

TGFbeta Induces Bim mRNA Expression-- To determine whether the up-regulation of Bim expression by TGFbeta 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 TGFbeta addition. In particular, the largest transcript (Fig. 5A, upper arrow) is induced greater than 3-fold following a 4-8-h TGFbeta treatment (Fig. 5B). The lower, most prominent transcript (Fig. 5A, bottom arrow), did not show any significant induction by TGFbeta . While Fig. 5A demonstrates that TGFbeta treatment induced the expression of several other less prominent transcripts, these transcripts were not reliably observed by Northern blot analysis.


View larger version (52K):
[in this window]
[in a new window]
 
Fig. 5.   TGFbeta induces Bim mRNA expression. A, Northern analysis of Bim mRNA levels. Smad3D cells were treated in the absence (Cont.) or presence of TGFbeta for up to 8 h. Poly(A)+ 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 beta -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 beta -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 beta -actin transcript. The ratio obtained for the untreated control (Cont.) sample was set at 100%, and the ratios obtained for the TGFbeta -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 TGFbeta -induced Bim protein expression. Smad3D cells were treated in the absence (-) or presence (+) of TGFbeta for 8 h. During the last 4 h of TGFbeta treatment, either cycloheximide (10 µg/ml) or actinomycin D (0.5 µg/ml) was also present. Whole cell lysates were prepared, and 50-µg aliquots analyzed for the presence of Bim, Bcl-2, and Hsp-90 by Western blotting.

In order to determine whether TGFbeta -induced Bim protein expression required new protein synthesis or mRNA transcription, S3D cells were co-incubated with cycloheximide or actinomycin D, respectively, during TGFbeta treatment. As shown in Fig. 5C, both cycloheximide and actinomycin D treatment inhibited Bim protein expression induced by TGFbeta . 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 TGFbeta -induced Bim expression occurs through a transcriptional mechanism.

TGFbeta 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 TGFbeta treatment. We performed immunoblot analysis on subcellular fractions of S3D cells treated in the absence or presence of TGFbeta for 24 h. As shown in Fig. 6A, TGFbeta 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 TGFbeta and again little change in Bcl-2 levels in the absence or presence of TGFbeta . 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.


View larger version (43K):
[in this window]
[in a new window]
 
Fig. 6.   TGFbeta 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 (-) or presence (+) of TGFbeta 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 µg), mitochondrial fraction (Mito., 50 µg) and cytosolic fraction (Cyto., 100 µg) 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 TGFbeta for up to 24 h, and whole cell lysates prepared. Bim protein was immunoprecipitated from 500 µg 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 µg) were directly analyzed by Western blotting for the presence of Bcl-2 (middle blot) and Hsp-90 (lower blot). C, mitochondrial depolarization induced by TGFbeta . Smad3D cells were incubated in the absence (Control) and presence of TGFbeta 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 TGFbeta samples.

Since TGFbeta promotes accumulation of Bim in the mitochondria, we wished to determine whether TGFbeta could also promote heterodimerization of Bim with Bcl-2. The results of Fig. 6B demonstrate by co-immunoprecipitation analysis that TGFbeta 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 TGFbeta 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 TGFbeta stimulation alters mitochondrial membrane potential in S3D cells. We used the lipophilic cationic dye DiOC6 (3) to stain mitochondria and monitored mitochondrial membrane depolarization by FACScan analysis. The individual histograms shown to the left in Fig. 6C demonstrate that TGFbeta 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 TGFbeta 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 TGFbeta -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 alpha -CD40 antibody is able to rescue or abrogate the apoptotic effects of TGFbeta in S3D cells. We next determined whether CD40 rescue of TGFbeta -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 alpha -CD40 and TGFbeta inhibits the induction of Bim protein mediated by TGFbeta . The induction of Bim protein following a 6- and 24-h TGFbeta treatment is inhibited to near basal levels following co-stimulation with alpha -CD40. Furthermore, the results of Fig. 7D demonstrate that the TGFbeta -induced complex formation between Bim and Bcl-2 following a 6- and 24-h TGFbeta treatment is inhibited by alpha -CD40. These data demonstrate that CD40 stimulation can rescue Smad3-overexpressing WEHI 231 cells from TGFbeta -mediated apoptosis and suggest that inhibition of Bim protein induction by CD40 stimulation may represent a mechanism by which pro-survival cytokines suppress the TGFbeta apoptotic response.


View larger version (48K):
[in this window]
[in a new window]
 
Fig. 7.   CD40 antagonizes TGFbeta -mediated events. A, CD40 rescue of TGFbeta -induced apoptosis. Smad3D cells were untreated (Cont.) or treated for 24 h with TGFbeta alone, CD40 alone or both TGFbeta 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 TGFbeta -induced DNA ladder formation. Smad3D cells were untreated (Cont.) or treated overnight with TGFbeta alone, CD40 alone, or both TGFbeta 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 TGFbeta -induced Bim protein expression. Smad3D cells were untreated (Cont.) or treated for 6 or 24 h with TGFbeta alone, CD40 alone, or both TGFbeta and CD40. Whole cell lysates were prepared and 50-µg aliquots were analyzed by Western blotting for the presence of Bim (upper blot) or Hsp-90 (lower blot). D, CD40 blocks TGFbeta -induced Bim/Bcl-2 binding. Lysates (500 µg) 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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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(-/-) 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 TGFbeta induces the expression of Bim in 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-XL (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 pro-survival 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 up-regulation 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, TGFbeta , 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 TGFbeta concomitant with its rescue of the cells from TGFbeta -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 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 TGFbeta -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 TGFbeta -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 TGFbeta -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 dominant-negative forms of Smad3 inhibit TGFbeta induced apoptotic cell death in Hep3B cells (63). More recently, TGFbeta -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 TGFbeta 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 TGFbeta that is regulated in a Smad-dependent manner. Overexpression of Smad3 results in a sensitization of WEHI 231 B lymphocytes to TGFbeta -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 TGFbeta -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, TGFbeta transcriptional responses (64-66). Computer analysis of the 5' 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 TGFbeta -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 transcription factors as a convergence point between the TGFbeta /Smad3-mediated cell death pathway and the CD40-mediated survival pathway in future studies.

    ACKNOWLEDGEMENTS

We thank Dr. Steve Ledbetter at Genzyme Inc. for generous provision of TGFbeta 2 and Dr. Edward Leof at the Mayo Clinic (Rochester, MN) for the phospho-Smad3 antibody. We also thank Cathy Stanko for assistance with the mitochondrial depolarization studies.

    FOOTNOTES

* This work was supported by Grants CA80095 and CA55536 from the NCI, National Institutes of Health (to P. H. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Cell Biology, NC-1, The Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195. Tel.: 216-445-9750; Fax: 216-445-7855; E-mail: howep@ccf.org.

Published, JBC Papers in Press, March 13, 2003, DOI 10.1074/jbc.M211958200

    ABBREVIATIONS

The abbreviations used are: TGFbeta , transforming growth factor beta ; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; Bim, BCL-2-interacting mediator of cell death; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling; FACS, fluorescence-activated cell sorter; ELISA, enzyme-linked immunosorbent assay.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Massague, J. (1998) Annu. Rev. Biochem. 67, 753-791[CrossRef][Medline] [Order article via Infotrieve]
2. Piek, E., Heldin, C.-H., and Dijke, P. T. (1999) FASEB J. 13, 2105-2124[Abstract/Free Full Text]
3. Wrana, J. L. (2000) Cell 100, 189-192[Medline] [Order article via Infotrieve]
4. Massagué, J., and Chen, Y-G. (2000) Genes Dev. 14, 627-644[Free Full Text]
5. Tsukazaki, T., Chiang, T. A., Davison, A. F., Attisano, L., and Wrana, J. L. (1998) Cell 95, 779-791[Medline] [Order article via Infotrieve]
6. Miura, S., Takeshita, T., Asao, H., Kimura, Y., Murata, K., Sasaki, Y., Hanai, J-I., Beppu, H., Tsukazaki, T., Wrana, J. L., Miyazono, K., and Sugamura, K. (2000) Mol. Cell. Biol. 20, 9346-9355[Abstract/Free Full Text]
7. Hocevar, B. A., Smine, A., Xu, X-X., and Howe, P. H. (2001) EMBO J. 20, 2789-2801[Abstract/Free Full Text]
8. Morris, S. M., and Cooper, J. A. (2001) Traffic 2, 111-123[CrossRef][Medline] [Order article via Infotrieve]
9. Yan, Z., Winawer, S., and Friedman, E. (1994) J. Biol. Chem. 269, 13231-13237[Abstract/Free Full Text]
10. Hartsough, M. T., and Mulder, K. M. (1995) J. Biol. Chem. 270, 7117-7124[Abstract/Free Full Text]
11. Atfi, A., Djelloul, S., Chastre, E., Davis, R., and Gespach, C. (1997) J. Biol. Chem. 272, 1429-1432[Abstract/Free Full Text]
12. Frey, R. S., and Mulder, K. M. (1997) Cancer Res. 57, 628-633[Abstract]
13. Hocevar, B. A., Brown, T. L., and Howe, P. H. (1999) EMBO J. 18, 1345-1356[Abstract/Free Full Text]
14. Engel, M. E., McDonnell, M. A., Law, B. K., and Moses, H. L. (1999) J. Biol. Chem. 274, 37413-37420[Abstract/Free Full Text]
15. Sano, Y., Harada, J., Tashiro, S., Gotoh-Mandeville, R., Maekawa, T., and Ishii, S. (1999) J. Biol. Chem. 274, 8949-8957[Abstract/Free Full Text]
16. Hanafusa, H., Ninomiya-Tsuji, J., Masuyama, N., Nishita, M., Fujisawa, J., Shibuya, H., Matsumoto, K., and Nishida, E. (1999) J. Biol. Chem. 274, 27161-27167[Abstract/Free Full Text]
17. Bakin, A. V., Tomlinson, A. K., Bhowmick, N. A., Moses, H. L., and Arteaga, C. L. (2000) J. Biol. Chem. 275, 36803-36810[Abstract/Free Full Text]
18. Engel, M. E., Datta, P. K., and Moses, H. L. (1998) J. Biol. Chem. 273, 9921-9926[Abstract/Free Full Text]
19. Mucsi, I., Skorecki, K. L., and Goldberg, H. J. (1996) J. Biol. Chem. 271, 16567-16572[Abstract/Free Full Text]
20. Bhowmick, N. A., Ghiassi, M., Bakin, A., Aakre, M., Lundquist, C. A., Engel, M. E., Arteaga, C. L., and Moses, H. L. (2001) Mol. Biol. Cell 12, 27-36[Abstract/Free Full Text]
21. Kyprianou, N., and Isaacs, J. T. (1989) Mol. Endocrinol. 3, 1515-1522[Abstract]
22. Hsing, A. Y., Kadomatsu, K., Bonham, M. J., and Danielpour, D. (1996) Cancer Res. 56, 5146-5149[Abstract]
23. Oberhammer, F. A., Pavelka, M., Sharma, S., Tiefenbacher, R., Purchio, A. F., Bursch, W., and Schulte-Hermann, R. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5408-5412[Abstract]
24. Lin, J-K., and Chou, C-K. (1992) Cancer Res. 52, 385-388[Abstract]
25. Chen, R-H., and Chang, T-Y. (1997) Cell Growth & Differ. 8, 821-827[Abstract]
26. Francis, J. M., Heyworth, C. M., Spooncer, E., Pierce, A., Dexter, T. M., and Whetton, A. D. (2000) J. Biol. Chem. 275, 39137-39145[Abstract/Free Full Text]
27. Arsura, M., Wu, M., and Sonenshein, G. E. (1996) Immunity 5, 31-40[Medline] [Order article via Infotrieve]
28. Brown, T. L., Patil, S., Cianci, C. D., Morrow, J. S., and Howe, P. H. (1999) J. Biol. Chem. 274, 23256-23262[Abstract/Free Full Text]
29. Patil, S., Wildey, G. M., Brown, T. L., Choy, L., Derynck, R., and Howe, P. H. (2000) J. Biol. Chem. 275, 38363-38370[Abstract/Free Full Text]
30. Perlman, R., Schiemann, W. P., Brooks, M. W., Lodish, H. F., and Weinberg, R. A. (2001) Nat. Cell Biol. 3, 708-714[CrossRef][Medline] [Order article via Infotrieve]
31. Jang, C-W., Chen, C-H., Chen, C-C., Chen, J-Y., Su, Y-H., and Chen, R-H. (2002) Nat. Cell Biol. 4, 51-58[CrossRef][Medline] [Order article via Infotrieve]
32. Larisch, S., Yi, Y., Lotan, R., Kerner, H., Eimerl, S., Parks, W. T., Gottfried, Y., Reffey, S. B., de Caestecker, M. P., Danielpour, D., Book-Melamed, N., Timberg, R., Duckett, C. S., Lechleider, R. J., Steller, H., Orly, J., Kim, S-J., and Roberts, A. B. (2000) Nat. Cell Biol. 2, 915-921[CrossRef][Medline] [Order article via Infotrieve]
33. Kim B-C, Mamura, M., Choi, K. S., Calabretta, B., and Kim, S-J. (2002) Mol. Cell. Biol. 22, 1369-1378[Abstract/Free Full Text]
34. O'Connor, L., Strasser, A., O'Reilly, L. A., Hausmann, G., Adams, J. M., Cory, S., and Huang, D. C. S. (1998) EMBO J. 17, 384-395[Abstract/Free Full Text]
35. Zamzani, N., Metivier, D., and Kroemer, G. (2000) Methods Enzymol. 322, 209-213
36. Dijkers, P. F., Medema, R. H., Lammers, J-W. J., Koenderman, L., and Coffer, P. J. (2000) Curr. Biol. 10, 1201-1204[CrossRef][Medline] [Order article via Infotrieve]
37. Marani, M., Tenev, T., Hancock, D., Downward, J., and Lemoine, N. R. (2002) Mol. Cell. Biol. 22, 3577-3589[Abstract/Free Full Text]
38. Mami, U., Miyashita, T., Shikima, Y., Tadokoro, K., and Yamada, M. (2001) FEBS Lett. 509, 135-141[CrossRef][Medline] [Order article via Infotrieve]
39. Puthalakath, H., Huang, D. C., O'Reilly, L. A., King, S. M., and Strasser, A. (1999) Mol. Cell 3, 287-296[Medline] [Order article via Infotrieve]
40. Sugiyama, T., Shimizu, S., Matsuoka, Y., Yoneida, Y., and Tsujimoto, Y. (2002) Oncogene 21, 4944-4956[CrossRef][Medline] [Order article via Infotrieve]
41. Vander Heiden, M. G., and Thompson, C. B. (1999) Nat. Cell Biol. 8, E209-216[CrossRef]
42. Kelekar, A., and Thompson, C. B. (1998) Trends Biochem. Sci. 8, 324-330[CrossRef]
43. O'Reilly, L. A., Cullen, L., Visvader, J., Lindeman, G. J., Print, C., Bath, M. L., Huang, D. C. S., and Strasser, A. (2000) Am. J. Pathol. 157, 449-461[Abstract/Free Full Text]
44. Zong, W-X., Lindsten, T., Ross, A. J., MacGregor, G. R., and Thompson, C. B. (2001) Genes Dev. 15, 1481-1486[Abstract/Free Full Text]
45. Harris, C. A., and Johnson, E. M. (2001) J. Biol. Chem. 276, 37754-37760[Abstract/Free Full Text]
46. Bouillet, P., Purton, J. F., Godfry, D. L., Zhang, L-C., Coultas, L., Puthalakath, H., Pellegrini, M., Cory, S., Adams, J. M., and Strasser, A. (1999) Science 286, 1735-1738[Abstract/Free Full Text]
47. Bouillet, P., Purton, J. F., Godfrey, D. I., Zhang, L-C., Coultas, L., Puthalakath, H., Pellegrini, M., Cory, S., Adams, J. M., and Stresser, A. (2002) Nature 415, 922-926[CrossRef][Medline] [Order article via Infotrieve]
48. Puthalakath, H., and Stresser, A. (2002) Cell Death Differ. 9, 505-512[CrossRef][Medline] [Order article via Infotrieve]
49. Zha, J., Harada, H., Yang, E., Jockel, J., and Korsmeyer, S. J. (1996) Cell 87, 619-628[Medline] [Order article via Infotrieve]
50. Datta, S. R., Dudek, H., Tao, X., Masters, S., Fu, H., Gotoh, Y., and Greenberg, M. E. (1997) Cell 91, 231-241[Medline] [Order article via Infotrieve]
51. del Peso, L., Gonzalez-Garcia, M., Page, C., Herrera, R., and Nunez, G. (1997) Science 278, 687-689[Abstract/Free Full Text]
52. Li, H., Zhu, H., Xu, C-J., and Yuan, J. (1998) Cell 94, 491-501[Medline] [Order article via Infotrieve]
53. Shinjyo, T., Kuribara, R., Inukai, T., Hosoi, H., Knioshita, T., Miyajima, A., Houghton, P. J., Look, T. A., Ozawa, K., and Inaba, T. (2001) Mol. Cell. Biol. 21, 854-864[Abstract/Free Full Text]
54. Putcha, G. V., Moulder, K. L., Golden, J. P., Bouillet, P., Adams, J. A., Strasser, A., and Johnson, E. M. (2001) Neuron 29, 615-628[Medline] [Order article via Infotrieve]
55. Whitfield, J., Neame, S. J., Paquet, L., Bernard, O., and Ham, J. (2001) Neuron 29, 629-643[Medline] [Order article via Infotrieve]
56. Stahl, M., Dijkers, P. F., Kops, G. J. P. L., Lens, S. M. A., Coffer, P. J., Burgering, B. M. T., and Medema, R. H. (2001) J. Immunol. 168, 5024-5031
57. Dijkers, P. F., Birkenkamp, K. U., Lam, E. W.-F., Thomas, N. S. B., Lammers, J-W. J., Koenderman, L., and Coffer, P. J. (2002) J. Cell Biol. 156, 531-542[Abstract/Free Full Text]
58. Yanagisawa, K., Osada, H., Masuda, A., Kondo, M., Saito, T., Yatabe, Y., Takagi, K., and Takahashi, T. (1998) Oncogene 17, 1743-1747[CrossRef][Medline] [Order article via Infotrieve]
59. Brodin, G., ten Dijke, P., Funa, K., Heldin, C. -H., and Landstrom, M. (1999) Cancer Res. 59, 2731-2738[Abstract/Free Full Text]
60. Ishisaki, A., Yamato, K., Nakao, A., Nonaka, K., Ohguchi, M., ten Dijke, P., and Nishihara, T. (1998) J. Biol. Chem. 273, 24293-24296[Abstract/Free Full Text]
61. Ishisaki, A., Yamato, K., Hashimoto, S., Nakao, A., Tamaki, K., Nonaka, K., ten Dijke, P., Sugino, H., and Nishihara, T. (1999) J. Biol. Chem. 274, 13637-13642[Abstract/Free Full Text]
62. Curnock, A. P., and Knox, K. A. (1998) Cell. Immunol. 187, 87-97
63. Yamamura, Y., Hua, X., Bergelson, S., and Lodish, H. F. (2000) J. Biol. Chem. 275, 36295-36302[Abstract/Free Full Text]
64. Chen, X., Weisberg, E., Fridmacher, V., Watanabe, M., Naco, G., and Whitman, M. (1997) Nature 389, 85-89[CrossRef][Medline] [Order article via Infotrieve]
65. Labbé, E., Silvestri, C., Hoodless, P. A., Wrana, J. L., and Attisano, L. (1998) Mol. Cell 2, 109-120[Medline] [Order article via Infotrieve]
66. Yeo, C. Y., Chen, X., and Whitman, M. (1999) J. Biol. Chem. 274, 26584-26590[Abstract/Free Full Text]
67. Bouillet, P., Zhang, L. C., Huang, D. C. S., Webb, G. C., Bottema, C. D. K., Shore, P., Eyre, H. J., Sutherland, G. R., and Adams, J. M. (2001) Mamm. Genome 12, 163-168[CrossRef][Medline] [Order article via Infotrieve]
68. Fruman, D. A., Snapper, S. B., Yballe, C. M., Davidson, L., Yu, J. Y., Alt, F. W., and Cantley, L. C. (1999) Science 283, 393-396[Abstract/Free Full Text]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.