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J. Biol. Chem., Vol. 275, Issue 46, 36295-36302, November 17, 2000

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Critical Role of Smads and AP-1 Complex in Transforming Growth Factor--dependent Apoptosis^*

Yasuko Yamamura^§¶, Xianxin Hua, Svetlana Bergelson, and Harvey F. Lodish^**

From the  Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, the ^** Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, and the ^§ Department of Retroviral Regulation, Tokyo Medical and Dental University Medical Research Division, Tokyo 113-8519, Japan

Received for publication, July 8, 2000, and in revised form, August 6, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Transforming growth factor-1 (TGF-1) induces not only cell growth inhibition but also apoptosis in hepatocytes, myeloid cells, and epithelial cells. Although Smad proteins are identified as key signal transducers in TGF-1-dependent growth inhibition, their roles in the induction of apoptosis are unclear. We show here that both Smad proteins and AP-1 complex are involved in TGF-1 signaling for apoptosis. Overexpression of a dominant-negative Smad3 mutant or Smad7, both of which impair Smad-mediated signal transduction, inhibits TGF-1-dependent apoptosis. Only the JunD·FosB form of the AP-1 complex is markedly activated during TGF-1-dependent apoptosis. FosB substantially enhances Smad3·Smad4-dependent transcription, and dominant-negative FosB blocks TGF-1-dependent apoptosis but not growth inhibition. Expression of JunD·FosB enhances induction of apoptosis by TGF-1. Moreover, JunD·FosB binds to the 12-O-tetradecanoyl-13-acetate-responsive gene promoter element and recruits Smad3·Smad4 to form a multicomponent complex. These results suggest that Smad proteins and AP-1 complex synergize to mediate TGF-1-dependent apoptosis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The transforming growth factor- (TGF-)¹ superfamily consists of a large number of cytokines including TGF-s, bone morphogenetic proteins, and activins, which exert a wide range of biological functions including cell proliferation, differentiation, and apoptosis (1, 2). TGF-1, the founding member of the superfamily, is well known as a negative regulator of cell growth. A variety of cell types express the TGF- receptors, and their cell growth is inhibited by TGF-1. Its apoptotic activity is also shown in specific cell types such as hepatocytes, myeloid cells, and epithelial cells. TGF-1 promotes apoptosis not only in a hepatoma cell line (3) but also in hepatocytes in regressing liver (4) and in cirrhotic liver (5). TGF-1 is implicated in the induction of apoptosis in prostatic epithelial cells after castration and is used to treat advanced prostatic tumor (6). Thus, TGF-1-dependent apoptosis plays an essential role in eliminating abnormal cells from normal tissues in vivo. On the other hand, myeloid cell development is known to be under the control of apoptosis induced by TGF-1 (7). These observations suggest that TGF-1 exerts two distinct biological activities: inhibition of cell proliferation and induction of apoptosis.

Recent studies demonstrate that Smad proteins transmit the signals of the TGF- superfamily members from their cell surface receptors to the nucleus (1, 2). Upon ligand activation of the receptor complex, receptor-regulated Smads become serine-phosphorylated by the activated receptors and associate with the common Smad4 to form a hetero-oligomeric complex. The complex then translocates to the nucleus, where it binds to DNA and regulates transcription of specific genes. The Smad2·Smad4 and Smad3·Smad4 complexes have been demonstrated to be responsible for TGF-1-dependent transcriptional activation of reporter constructs and growth inhibition. Although overexpression of Smad4 is suggested to induce apoptosis in MDCK canine epithelial cells (8), TGF-1-induced overexpression of Smad7, which inhibits TGF-1 signaling mediated by Smad2·Smad4 and Smad3·Smad4 complexes (9, 10), leads to apoptosis in human prostatic carcinoma cells (11). The role of Smad proteins is still obscure in TGF-1-dependent apoptosis.

TGF-1 has been reported to immediately and transiently up-regulate mRNA levels of genes of AP-1 components such as junB (12) and c-jun (13), but the role of these up-regulated AP-1 components in TGF-1-dependent growth inhibition and apoptosis is unknown. The promoters of TGF-1-responsive genes like plasminogen activator inhibitor-1 (PAI-1) and c-jun contain AP-1 binding sites. Mutation in these AP-1 binding sites, which impairs binding of the AP-1 complex, inhibits transcriptional activation of these promoters by TGF-1 (14, 15). These findings suggest that Smad proteins and the AP-1 complex synergize to activate the TGF-1-responsive promoters. Recent studies indicate that Smad3 directly binds c-Jun and c-Fos of the AP-1 complex (16) and that both Smad3 and Smad4 bind all three Jun proteins, c-Jun, JunB, and JunD (17). However, involvement of the AP-1 complex has not been demonstrated either in growth inhibition or in apoptosis induced by TGF-1.

The AP-1 complex appears to be involved in cell proliferation and survival, and a role of this multicomponent complex is also suggested in apoptosis of some cell types (18). A dominant-negative mutant form of c-Jun, a central component of the AP-1 complex, protects sympathetic neurons from apoptosis induced by nerve growth factor deprivation (19). Activation of c-Jun by c-Jun N-terminal kinase is required for induction of Fas/Fas ligand (FasL)-mediated apoptosis in Jurkat T cells and PC12 cells (20, 21). However, the AP-1 complex does not participate in apoptosis induced by TNF- or cross-linking of Fas with agonistic anti-Fas antibody (22, 23). It is still unknown whether the AP-1 complex is actually involved in TGF-1-dependent apoptosis.

Our present results show that TGF-1 activates Smad proteins and the AP-1 complex (JunD·FosB) and that overexpression of their dominant-negative forms inhibits TGF-1-dependent apoptosis. Furthermore, we find that overexpression of FosB enhances Smad-dependent transcription of the TGF-1-responsive reporter. JunD·FosB recruits Smad3·Smad4 to form the AP-1·Smad complex that binds to the AP-1 binding site, 12-O-tetradecanoyl-13-acetate-responsive gene promoter element. These results show that both Smad proteins and AP-1 complex play a critical role in TGF-1-dependent apoptosis.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture and Transfection-- M1 and Hep3B cell lines were purchased from the American Type Culture Collection (ATCC). M1 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. Hep3B cells were cultured in minimum essential medium supplemented with 10% fetal bovine serum. Transient and stable transfection was performed using the calcium phosphate DNA precipitation method and SuperFect Transfection (Qiagen), respectively. Stable transfectants were selected with G418 (Life Technologies, Inc.), hygromycin-B (Life Technologies, Inc.), or Zeocin (Invitrogen).

Plasmid Construction-- A FLAG epitope was introduced by polymerase chain reaction into the N terminus of Smad3 cDNA from pRK5-FLAG-hMAD-3 (24). A cDNA for FLAG-tagged Smad3 mutant (FLAG-Smad3D) in which three C-terminal serine residues were changed to alanine residues was generated using the QuikChange site-directed mutagenesis kit (Stratagene) and was fully sequenced. A HA epitope was introduced by polymerase chain reaction into the N terminus of Smad4 cDNA from pCMV5B-FlagMADR4 plasmid (25). A HA epitope was also introduced to c-Jun cDNA from JAC.1 plasmid (26) by subcloning c-Jun cDNA into pJ3H vector (ATCC). A FLAG epitope was introduced by polymerase chain reaction into the N terminus of JunD cDNA from XHJ-12.4 plasmid (ATCC) and was fully sequenced. An Myc epitope was introduced to cDNAs for FosB and truncated FosB (FosB), encoding the first 237 amino acids of FosB from FosB2L plasmid (ATCC) by subcloning the cDNAs into pcDNA3.1/Myc-His vector (Invitrogen). The above cDNAs were further subcloned into pcDNA3.1(+) and pIND (Invitrogen) vectors. pVgRXR vector was purchased from Invitrogen.

Assays for Cell Proliferation and Apoptosis-- Cell proliferation was estimated by a colorimetric assay using the tetrazolium salt 4-[3-(4-lodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) (Roche Molecular Biochemicals) according to manufacturer's instructions. Viable cell number was determined by using the trypan blue dye exclusion method. For DNA fragmentation assays, cells were lysed in lysis buffer (10 mM Tris, pH 7.5, 10 mM EDTA, 0.2% Triton X-100). The cell lysates were centrifuged, and supernatants were extracted with phenol/chloroform. DNA was precipitated with ethanol and analyzed by electrophoresis on a 2% agarose gel. Apoptosis was quantified by the cell death detection ELISA assay (Roche Molecular Biochemicals), which measures the presence of cytoplasmic histone-associated mono- and oligonucleosomes as a result of apoptosis.

[³⁵S]Methionine Labeling and Immunoprecipitation-- Cells were labeled with [³⁵S]methionine and lysed. The cell lysates were subjected to immunoprecipitation with an appropriate antibody. Immunoprecipitates were analyzed by SDS-PAGE and visualized by autoradiography.

Western Blotting-- Whole cell lysates were resolved on SDS-PAGE and transferred to Immobilon-P membrane (Millipore). Immunoblots were probed with an appropriate first antibody and a horseradish peroxidase-conjugated second antibody. Immunocomplexes were detected by Renaissance enhanced chemiluminescence (PerkinElmer Life Sciences).

Luciferase Assays-- Cells were transiently transfected with an indicated reporter construct and an internal control pRL-TK vector (Promega). Luciferase activity was measured 20 h later using the dual luciferase assay system (Promega) in MicroLumat LB96P luminometer (EG & G Berthold). The luciferase activity was normalized for transfection efficiency using the Renilla luciferase activity from pRL-TK.

Electrophoretic Mobility Shift Assay (EMSA)-- Nuclear extracts were prepared, and EMSA was performed as described previously (27). Complementary oligonucleotides containing the 12-O-tetradecanoyl-13-acetate-responsive gene promoter element (TRE) (5'-CGCTTGATGAGTCAGCCGGAA-3' and its complementary strand) were labeled with [-³²P]ATP using T4 polynucleotide kinase (New England Biolabs). A radiolabeled oligonucleotide probe (2 × 10⁴ cpm) and 10 µg of nuclear extracts were incubated for 15 min at room temperature. DNA binding complexes were separated on 5% polyacrylamide gels in 0.5× TBE and detected by autoradiography. For supershift experiments, the nuclear extracts were pre-incubated with an appropriate antibody. For competition experiments, the extracts were incubated with a 100-fold molar excess of unlabeled complementary oligonucleotides.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

TGF-1-dependent Growth Inhibition and Apoptosis-- Although lower concentrations (less than 50 pM) of TGF-1 induces only cell growth inhibition, more than 100 pM of TGF-1 induces not only growth inhibition but also apoptosis in a murine myeloid cell line M1 (28) and a human hepatoma cell line Hep3B (3). We first examined the kinetics of TGF-1-dependent cell growth inhibition and apoptosis in M1 and Hep3B cells. A colorimetric cell proliferation assay showed that cell growth was markedly inhibited after 24 h of stimulation by 10 and 400 pM TGF-1 (Fig. 1A). The level of growth inhibition was not increased by additional TGF-1 stimulation. In contrast, apoptosis was induced after 24 h by 400 pM TGF-1 (Fig. 1, B, C, and D) but not by 10 pM TGF-1 (Fig. 1D). The level of apoptosis was increased during 72 h after the TGF-1 stimulation. Similar apoptotic response was also obtained when cells were treated with 100 pM TGF-1 (data not shown).

View larger version (24K): [in this window] [in a new window]   Fig. 1.   TGF-1-dependent growth inhibition and apoptosis. A, M1 and Hep3B cells were cultured for indicated times with 10 pM (hatched bars), 400 pM (filled bars), or without (open bars) TGF-1. Their cell growth was estimated by colorimetric WST-1 assays. B, cells were cultured with (closed circles) or without (open circles) 400 pM TGF-1. The percentage of viable cells was determined using trypan blue dye exclusion assays. C, cells were cultured with (+) or without () 400 pM TGF-1, and their DNA fragmentation was analyzed by agarose gel electrophoresis. D, cells were cultured with 10 pM (hatched bars), 400 pM (filled bars), or without (open bars) TGF-1, and their apoptosis was assessed by cell death detection ELISA assays.

A Dominant-negative Smad3 Mutant and Inhibitory Smad7 Block TGF-1-dependent Apoptosis-- To investigate whether Smad proteins are directly involved in TGF-1 signaling for apoptosis, we expressed dominant-negative mutant Smad3 or inhibitory Smad7 in Hep3B cells. As shown in Fig. 2A, each stable transfectant expressed the FLAG-tagged dominant-negative Smad3 mutant (FLAG-Smad3D) or the FLAG-tagged Smad7 (FLAG-Smad7). Expression of FLAG-Smad3D and FLAG-Smad7 significantly inhibited the apoptosis induced by TGF-1 (Fig. 2B), the latter (50% inhibition) more effectively than the former (36% inhibition). Dominant-negative mutant forms of Smad2 and Smad3 synergize in inhibition of apoptosis, and their effect (52% inhibition) is almost the same as that of FLAG-Smad7 (data not shown). These results suggest that the Smad complexes play a critical role in TGF-1-dependent apoptosis. Consistent with previous reports, expression of FLAG-Smad3D and FLAG-Smad7 diminished TGF-1-dependent growth inhibition (Fig. 2C) and transcriptional activation of p3TP-Lux reporter construct, which contains the TGF-1-responsive PAI-1 promoter (Fig. 2D).

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Effects of dominant-negative Smad3 and inhibitory Smad7 on TGF-1-dependent apoptosis, growth inhibition, and transcriptional activation. A, expression of FLAG-tagged Smad proteins in Hep3B-derived transfectant clones. Total cell lysates were analyzed by SDS-PAGE. Western blotting was performed using anti-FLAG M2 monoclonal antibody (Sigma). B and C, cells were cultured for 72 h with (filled bars) or without (open bars) 400 pM TGF-1. Apoptosis was assessed by cell death detection ELISA assays (B). Cell growth was estimated by colorimetric WST-1 assays (C). D, cells were transfected with the p3TP-Lux reporter construct and then incubated with (filled bars) or without (open bars) 400 pM TGF-1 for 20 h. The relative luciferase activity was measured in cell lysates. pcDNA is a cell clone expressing pcDNA3.1 empty vector, Smad3D is a cell clone expressing a FLAG-Smad3 mutant (FLAG-Smad3D), and Smad7 is a cell clone expressing FLAG-Smad7.

TGF-1-dependent Apoptosis Is Accompanied by Marked Activation of the AP-1 Complex-- Although the AP-1 complex has been shown to mediate apoptosis (19-21), its role remains unidentified in TGF-1-dependent apoptosis. We examined the kinetics of the AP-1 activity in M1 cells following the TGF-1 treatment. EMSA was performed using an oligonucleotide probe containing the consensus AP-1 binding site, TRE (29). When the cells underwent apoptosis following the stimulation of 400 pM TGF-1, DNA binding activity of AP-1 was markedly activated, and its activation lasted beyond 72 h (Fig. 3A). A 100-fold molar excess of an unlabeled TRE probe prevented the DNA binding activity of AP-1 induced by 400 pM TGF-1, indicating the binding is specific for the AP-1 binding site (Fig. 4A). Similar activation of AP-1 DNA binding was obtained when cells were stimulated with 100 pM TGF-1 (data not shown). In contrast, the AP-1 activity was transiently increased after 12 h but returned to a background level 24 h after 10 pM TGF-1 treatment, which induces growth arrest but not apoptosis (Fig. 3B). Both 10 and 400 pM TGF-1 induced similar weak and transient increases of the AP-1 activity in HepG2 cells, which do not respond to the TGF-1 treatment to induce apoptosis (data not shown).

View larger version (82K): [in this window] [in a new window]   Fig. 3.   Kinetics of AP-1 activation by TGF-1. M1 cells were unstimulated (0 h) or stimulated with 400 pM (A) or 10 pM (B) TGF-1 for the indicated times. Nuclear extracts were prepared and analyzed by EMSA with end-labeled TRE probe.

View larger version (64K): [in this window] [in a new window]   Fig. 4.   Identification of the Jun and Fos proteins involved in the AP-1 complex activated by TGF-1. M1 and Hep3B cells were unstimulated (0 h) or stimulated with 400 pM TGF-1 for the indicated times. Nuclear extracts were prepared and incubated in the absence (Control) or presence of a 100-fold molar excess of unlabeled TRE probe (Competitor) or 1 µg of an antibody (Santa Cruz) against each Jun protein (A) or Fos protein (B). The nuclear extracts were then analyzed by EMSA with end-labeled TRE probe. -c-Jun is an anti-c-Jun antibody, -JunB is an anti-JunB antibody, -JunD is an anti-JunD antibody, -c-Fos is an anti-c-Fos antibody, -FosB is an anti-FosB antibody, -Fra-1 is an anti-Fra-1 antibody, and -Fra-2 is an anti-Fra-2 antibody.

JunD·FosB Is the AP-1 Form Activated by TGF-1-- The AP-1 complexes consist of Jun and Fos proteins. Three Jun proteins, c-Jun, JunB, and JunD, homodimerize or heterodimerize with any of four Fos proteins, c-Fos, FosB, Fra-1, and Fra-2 (18). We performed supershift assays using antibodies against Jun (Fig. 4A) and Fos (Fig. 4B) proteins. Anti-JunD and anti-FosB antibodies caused a supershift of the TGF-1-activated AP-1 complex derived from M1 and Hep3B cells, indicating the presence of JunD and FosB in the complex. No supershift was seen with anti-JunB, anti-c-Fos, anti-Fra-1, or anti-Fra-2 antibody. Anti-c-Jun antibody slightly induced a supershift of the complex from Hep3B cells but not from M1 cells. Since Fos proteins neither homodimerize by themselves nor possess intrinsic specific DNA binding activity (18), these results indicate that JunD·FosB is the main form of the AP-1 complex activated by TGF-1. We next investigated the physical interaction between JunD·FosB and Smad proteins. JunD·FosB and Smad3·Smad4 are formed when each of their components is overexpressed in COS cells (30, 31). COS7 cells were cotransfected with expression vectors for JunD, FosB, FLAG-Smad3, and HA-Smad4. Using nuclear extracts derived from the COS7 cells, we performed EMSA with the TRE probe. As shown in Fig. 5, the TRE probe bound a complex from the COS7 lysate, and the binding was abolished by competition with a 100-fold molar excess of unlabeled TRE. All antibodies against FLAG and HA epitopes, JunD, and FosB caused a supershift of the TRE binding complex and shifted the complex to almost the same mobility, indicating that JunD, FosB, FLAG-Smad3, and HA-Smad4 are all present in the same TRE binding complex. Extracts from COS7 cells transfected only with the expression vectors for FLAG-Smad3 and HA-Smad4 did not yield the TRE binding complex (data not shown), whereas a previous study showed that Smad3 directly bound TRE (16). FosB is unable to bind TRE by itself (18). Together, these results indicate that the TRE complex is composed of JunD·FosB and Smad3·Smad4.

View larger version (50K): [in this window] [in a new window]   Fig. 5.   The TRE binding complex involves JunD·FosB and Smad3·Smad4. Supershift assays were performed with end-labeled TRE probe and nuclear extracts derived from COS7 cells transfected with expression plasmids for JunD, FosB, FLAG-Smad3, and HA-Smad4. A 100-fold molar excess of unlabeled probes (Competitor) or 1 µg of an antibody against JunD (JunD), FosB (FosB), a FLAG (FLAG), or a HA (HA) epitope (Roche Molecular Biochemicals) was included in the reaction as indicated. C, TRE binding complexes; SS, supershifted complexes.

FosB Enhances TGF-1- and Smad3·Smad4-dependent Transcription-- We next examined the effect of c-Jun, JunD, or FosB overexpression on transcription from the TGF-1-responsive p3TP-Lux reporter construct, which has the PAI-1 promoter element and three AP-1 binding sites. Hep3B cells were transiently cotransfected with p3TP-Lux and a control vector or an expression vector encoding c-Jun, JunD, or FosB. Overexpression of FosB, but not of c-Jun or JunD, yielded a 5-fold increase in TGF-1-dependent p3TP-Lux activity and slightly activated transcription of p3TP-Lux even without the TGF-1 stimulation (Fig. 6A). A truncated form of FosB, FosB, forms DNA binding heterodimers with the Jun proteins but lacks transcriptional activity (32). Overexpression of FosB neither enhanced TGF-1-dependent luciferase activity nor activated transcription from p3TP-Lux in the absence of TGF-1 (Fig. 6A). Moreover, we found that the FosB expression dramatically enhanced Smad3·Smad4-dependent activity of p3TP-Lux (Fig. 6B).

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Effect of FosB overexpression on TGF-1- and Smad-dependent transcription. A, Hep3B cells were transfected with pcDNA3.1 empty vector or an expression vector encoding c-Jun, JunD, FosB, or FosB together with the p3TP-Lux reporter construct. The cells were then incubated with (filled bars) or without (open bars) 400 pM TGF-1 for 20 h. The relative luciferase activity was measured in cell lysates. B, Hep3B cells were cotransfected with pcDNA3.1 empty vector or FosB expression vector in a combination of pcDNA3.1 empty vector (open bars) or Smad3 and Smad4 expression vectors (filled bars) together with the p3TP-Lux reporter construct. The relative luciferase activity was measured in cell lysates.

A Dominant-negative FosB Mutant Inhibits TGF-1-dependent Apoptosis-- The preceding results suggest that JunD·FosB interacts with Smad3·Smad4 and that FosB increases the transcriptional activity of Smad3·Smad4 that is involved in TGF-1 signaling for apoptosis. To further test whether an altered function of FosB interferes with TGF-1-dependent apoptosis, we established stable Hep3B cell clones that express different levels of FosB (Fig. 7A). 7 and 10 cell clones were resistant to TGF-1-dependent apoptosis, whereas 11 clone expressing only a trace of FosB was sensitive (Fig. 7B). Their resistance to TGF-1 depended on their expression levels of FosB. 7 clone expressing a higher level of FosB was much more resistant to apoptosis (67% inhibition of apoptosis) than 10 was (46% inhibition), whereas both were still sensitive to TGF-1-dependent growth inhibition (Fig. 7C). These results suggest that FosB participates in TGF-1 signaling for apoptosis but not growth inhibition in Hep3B cells.

View larger version (13K): [in this window] [in a new window]   Fig. 7.   Inhibition of TGF-1-dependent apoptosis by dominant-negative FosB (FosB). A, expression of Myc-tagged FosB in Hep3B-derived transfectant clones. Total cell lysates were analyzed by SDS-PAGE. Western blotting was performed using an anti-c-Myc 9E10 monoclonal antibody (Roche Molecular Biochemicals). B and C, cells were cultured for 72 h with (filled bars) or without (open bars) 400 pM TGF-1. Apoptosis was assessed by cell death detection ELISA assays (B). Cell growth was estimated by colorimetric WST-1 assays (C). pcDNA, a cell clone expressing pcDNA3.1 empty vector; 7, 10, and 11, cell clones expressing Myc-FosB.

Furthermore, we overexpressed JunD and FosB under the control of an ecdysone-inducible promoter in Hep3B cells and examined their effect on TGF-1-dependent apoptosis. Treatment of the JunD + FosB cell clone with ecdysone analog, muristerone A, increased expression of both FLAG-JunD and Myc-FosB (Fig. 8A), resulting in enhancement of TGF-1-dependent apoptosis (Fig. 8B).

View larger version (17K): [in this window] [in a new window]   Fig. 8.   JunD·FosB enhances induction of apoptosis by TGF-1. A, expression of FLAG-JunD and Myc-FosB is induced by muristerone A in Hep3B-derived transfectants. Cells were stimulated with (+) or without () muristerone A (MuriA). For detection of FLAG-JunD, cells were labeled with [35S]methionine. FLAG-JunD was immunoprecipitated with an anti-FLAG monoclonal antibody, analyzed by SDS-PAGE, and visualized by autoradiography. For detection of Myc-FosB, total cell lysates were analyzed by SDS-PAGE. Western blotting was performed with an anti-c-Myc monoclonal antibody to detect Myc-FosB. B, cells were cultured without muristerone A and TGF-1 (open bars), with 1 µM muristerone A (hatched bars), with 400 pM TGF-1 (gray bars), or with muristerone A and TGF-1 (closed bars) for 96 h. Apoptosis was assessed by cell death detection ELISA assays. pIND is a cell clone transfected with pIND empty vector and pVgRXR vector, and JunD + FosB is a cell clone transfected with pIND/Hygro/FLAG-JunD, pIND/Neo/Myc-FosB, and pVgRXR vectors.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

It is well established that Smad proteins are key components in TGF-1 signaling for growth inhibition and mesoderm formation (1, 2). We show here that not only Smad proteins but also AP-1 complex actually participate in TGF-1 signaling for apoptosis and that interference of the function of either Smad proteins or FosB component of the AP-1 complex markedly impairs TGF-1-dependent apoptosis.

Smad2 and Smad1/Smad5 have been shown to mediate activin and bone morphogenetic protein 2-dependent apoptosis of murine B cell hybridoma (33). Overexpression of Smad7 impairs phosphorylation of Smad proteins induced by activin or bone morphogenetic protein 2, resulting in inhibition of apoptosis. Although these observations indicate that Smad proteins are involved in activin or bone morphogenetic protein 2 signaling for apoptosis, their roles are unclear in TGF-1-dependent apoptosis. Overexpression of Smad4, but not Smad3, is proposed to mediate apoptosis in MDCK cells (8). However, TGF-1-dependent apoptosis of the cells is not indicated in this report. In fact, a recent report indicates that TGF-1 induces growth inhibition but not apoptosis in MDCK cells (34). Although Smad3 overexpression is shown to induce apoptosis in BEAS2B epithelial cells, TGF-1 stimulation is required to induce apoptosis in addition to Smad3 overexpression (35). We demonstrated here that TGF-1-dependent apoptosis was inhibited by expression of either dominant-negative mutant Smad3 or Smad7 (Fig. 2B), indicating that formation of the Smad complex is necessary for induction of apoptosis by TGF-1.

Although Smad proteins are suggested to cooperate with the AP-1 complex to regulate transcription of target genes, involvement of the AP-1 complex has not been demonstrated in cell growth inhibition or apoptosis induced by TGF-1. We found that only JunD and FosB, but not the other components of the AP-1 complex, are markedly induced during TGF-1-dependent apoptosis (Fig. 4, A and B). FosB enhanced Smad3·Smad4-dependent transcription (Fig. 6B), and its dominant-negative form blocked TGF-1-dependent apoptosis but not growth inhibition (Fig. 7, B and C). These results suggest that JunD·FosB activates transcription of a putative target gene for TGF-1, which is responsible for apoptosis. Previous reports have shown that the AP-1 complex directly binds FasL promoter and activates FasL expression, resulting in Fas/FasL-mediated apoptosis of Jurkat T cells and PC12 cells (20, 21). We, however, found that there was no expression of Fas in Hep3B cells, whereas FasL expression was increased by TGF-1 (data not shown). It remains to be determined which gene is involved in TGF-1-dependent apoptosis under the control of Smads and AP-1. Although c-Myc overexpression promotes apoptosis, c-Myc down-regulation also leads to apoptosis in some cell types. c-Myc is down-regulated by TGF-1 (36), and its promoter contains AP-1 binding site. Smads and AP-1 might recruit corepressors to block c-myc transcription to mediate TGF-1-dependent apoptosis.

Our present results suggest synergistic cooperation between Smads and the AP-1 complex in TGF-1-dependent apoptosis. Recently, a zinc finger protein, OAZ, was identified as a DNA binding factor that interacts with Smad1·Smad4 and Olf-1·EBF transcription factor using its different zinc finger domains to modulate transcriptional activity of distinct gene promoters (37). Similar to OAZ, Smads may coordinate distinct signals from multiple signaling cascades by interacting with specific partners. Indeed, Smad proteins have been shown to cooperate with a variety of transcription factors, AP-1, ATF2, FAST, and TFE3, and with nuclear oncoproteins, Evi-1, E1A, Ski, and SnoN, all of which repress TGF-1 signaling (38). Smad proteins may positively or negatively modify transcription of target genes through cooperation with their DNA binding partners to exert diverse biological activities.

Smad proteins are suggested to be tumor suppressors. Smad2 and Smad4 are functionally mutated in colorectal carcinoma (39) and pancreatic cancer (40), respectively. Smad3 knockout mice develop metastatic colorectal cancer (41). On the other hand, TGF-1 has been implicated as inducing apoptosis to eliminate abnormal cells from normal tissues such as the liver and prostate (4-6). Loss of sensitivity to the apoptotic effect of TGF-1 could contribute to the development of hepatocellular carcinoma and prostatic tumor. This is consistent with the fact that higher concentrations of TGF-1 at more than physiological concentration is required to induce apoptosis in the hepatoma and myeloma cell lines we used in our present study. An understanding of the cooperation between Smads and the AP-1 complex in TGF-1-dependent apoptosis could further show us the mechanisms underlying tumorigenesis.

	ACKNOWLEDGEMENTS

We thank Drs. J. L. Wrana, R. Derynck, D. Nathans, and M. Kawabata for providing the plasmid DNAs. TGF-1 was a generous gift from R & D Systems, Inc. We also thank Dr. Y. Ikawa for encouragement and support and Drs. R. Lin and M. Socolovsky for helpful discussions.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grant CA63260 (to H. F. L.).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.

^¶ Supported by a fellowship from the Research Training Program of the National Cancer Institute of the United States of America and the Japanese Foundation for Cancer Research of Japan. To whom correspondence should be addressed: Dept. of Retroviral Regulation, Tokyo Medical and Dental University Medical Research Division, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. Tel.: 81-3-5803-5161; Fax: 81-3-3814-7172; E-mail: yama.mbch@med.tmd.ac.jp.

Recipient of the Howard Temin Award and Burroughs Wellcome Career Development Award.

Published, JBC Papers in Press, August 14, 2000, DOI 10.1074/jbc.M006023200

	ABBREVIATIONS

The abbreviations used are: TGF-1, transforming growth factor-1; TRE, 12-O-tetradecanoyl-13-acetate-responsive gene promoter element; PAI-1, plasminogen activator inhibitor-1; FasL, Fas ligand; WST-1, 4-[3-(4-lodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate; EMSA, electrophoretic mobility shift assay; HA, hemagglutinin; ELISA, enzyme-linked immunosorbent assay; PAGE, polyacrylamide gel electrophoresis.

	REFERENCES

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