Tgf n Apoptosis

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Abstract Transforming growth factor-beta (TGF- ) is amultifunctional cytokine, whose numerous cell and tis-sue activities include cell-cycle control, the regulation of

early development, differentiation, extracellular matrixformation, hematopoesis, angiogenesis, chemotaxis, im-mune functions, and the induction of apoptosis. TGF- -mediated growth inhibition and apoptosis can be corre-lated with its function as a tumor suppressor. The apop-tosis-inducing capacity has been investigated in manycell types. Data from cell-culture experiments and in vi-vo studies argue for a pivotal role of TGF- -mediatedapoptosis in the maintenance of B- and T-cell homeosta-sis. The importance of TGF- in the control of liver cellapoptosis and cell death of prostate epithelial cells hasbeen confirmed in many studies. Inactivation of TGF- in animal models via a knockout approach or neutraliz-ing antibodies suggests that TGF- -mediated apoptosisplays an important part during tissue formation and re-modeling and during the phase of ontogenetic neurondeath. The molecular mechanisms involved in these pro-cesses seem to involve the activation of SMAD proteins.Many studies have described an interaction of TGF- with other signalinging cascades as exemplified by therequirement of AP1 transcription factor for the inductionof apoptosis in liver cells . The aim of this review is (1)to summarize and classify data in the TGF- apoptosisliterature with respect to the affected cell types, (2) toprovide insights into the intracellular mechanisms in-volved in TGF- -mediated apoptosis, and (3) to setTGF- -mediated apoptosis in a physiological context.

Keywords TGF- Apoptosis Regulation Caspase SMAD

Introduction

Transforming growth factor-beta (TGF- , also TGF- 1)was originally discovered by virtue of its capacity to in-duce anchorage-independent growth of normal rat kid-ney cells and fibroblast cell lines, i.e., to induce transfor-mation (Moses et al. 1981; Roberts et al. 1981). It soonbecame apparent that the biological activity of TGF- was not restricted to this effect on cell growth and that itwas instead multifunctional. The numerous cell and tis-sue roles of TGF- include cell-cycle control, the regula-tion of early development, differentiation, extracellularmatrix formation, hematopoesis, angiogenesis, chemo-taxis, and immune functions (Bttner et al. 2000; Dnkerand Krieglstein 2000; Lawrence 1996; Mummery 2001;Saltis 1996). TGF- represses growth of many epithelial

cell types, whereas the growth of mesenchymal cells isstimulated (Gold 1999). TGF- -mediated growth inhibi-tion and apoptosis can be correlated with its function asa tumor suppressor (Gold 1999; Hata et al. 1998). A sim-plified view of the TGF- signaling pathway is shown inFig. 1. TGF- binds to its membrane-bound receptors,TGF- receptors 1 and 2 (TGF RI, TGF RII), whichform a heterotetrameric complex. TGF RI is phosphory-lated by TGF RII in the so-called GS-domain. Upon thisactivating phosphorylation, TGF RI phosphorylates thereceptor-activated SMAD proteins (SMAD2 andSMAD3), which form a heteromeric complex with theco-SMAD SMAD4 and enter the nucleus for transcrip-tional regulation thereby involving other components of the transcriptional machinery. An autoregulatory feed-back loop is established by the induction of inhibitorySMAD proteins (SMAD6 or SMAD7) that prohibit theactivation of receptor-activated SMADs (the currrentview of the TGF- signal transduction pathway is bestreviewed by Massague 2000). In many human tumors,the components of the TGF- pathway are in some waydefective, either because of an inactivating mutationwithin the TGF- receptors (Larisch et al. 2000) or withregard to one of the downstream elements of the path-way, e.g., SMAD4/DPC4, which is important for intra-

Supported by the Deutsche Forschungsgemeinschaft

N. Schuster K. Krieglstein ( )Department of Anatomy and Cell Biology,Medical Faculty, University of Saarland,Building 61, 66421 Homburg/Saar, Germanye-mail: [email protected].: +49-6841-1626105, Fax: +49-6841-1626104

Cell Tissue Res (2002) 307:114DOI 10.1007/s00441-001-0479-6

R E V I E W

Norbert Schuster Kerstin Krieglstein

Mechanisms of TGF-

-mediated apoptosis

Received: 31 August 2001 / Accepted: 25 September 2001 / Published online: 8 November 2001 Springer-Verlag 2001


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cellular TGF- mediated signal transduction (Taketo andTakaku 2000). Although the apoptosis-inducing capacityof TGF- is well established in vivo and for a plethora of cell lines, we feel that there is major lack in summarizingthe various data for interpretation and in classifyingthem into a physiological context. The aim of this reviewis (1) to summarize and classify data in the TGF- apop-tosis literature with respect to the affected cell types, (2)

to provide insights into the intracellular mechanisms in-volved in TGF- mediated apoptosis, and (3) to set TGF--mediated apoptosis in a physiological context.

TGF--induced apoptosis

TGF- -induced apoptosis in cells of the immune system

TGF- induces growth arrest and apoptosis in lympho-cytes of human and mouse origin (Chaouchi et al. 1995;Kehrl et al. 1986; Lomo et al. 1995; Wahl et al. 1988).The mouse pre-B lymphoma cell line WEHI 231 re-

sponds to TGF- in a dose-dependent manner (Brown etal. 1998). Upon TGF- treatment, cell growth is inhibit-ed, but with increasing doses of TGF- , a substantialnumber of the cells die by apoptosis. A broad-spectrumcaspase inhibitor (BD-fmk), but not specific caspase in-hibitors, can completely block this apoptotic cell loss.Whereas cellular viability is maintained, the growth ar-rest persists, thereby distinguishing the growth inhibitoryeffects of TGF- from an apoptotic TGF- pathway. Inthe same cell line, TGF- - and IgM-induced apoptosiscan be inhibited by CD40 stimulation (Patil et al. 2000).Analysis of this phenomenon has revealed an upregula-tion of SMAD7 protein in response to CD40 stimulation.

SMAD7 is an inhibitory SMAD that blocks TGF- me-diated SMAD signaling by preventing the activatingphosphorylation of receptor-activated SMADs (Heldin etal. 1997; Massague 2000; Fig. 1). The activation of SMAD7 is dependent on NF B, as pathway-specific in-hibitors block the CD40-stimulated expression of SMAD7 (Patil et al. 2000). Further experiments demon-strating the importance of SMAD-dependent signaltransduction in this process have been performed by in-ducing the ectopic overexpression of either SMAD7 ordominant negative mutants of SMAD2 and SMAD3.Patil et al. (2000) have shown that interference with theSMAD pathway inhibits TGF- induced cell death in thepre-B lymphoma cell line WEHI 231, whereas IgM-in-duced apoptosis is not affected.

The induction of apoptosis has also been shown in twointerleukin-2-dependent T-cell lines, OVA-7 and CTLL-2(Weller et al. 1994). Cycloheximide prevents TGF- -in-duced apoptosis in CTLL-2 cells, but not in OVA-7 cells,indicating that protein biosynthesis is required in CTLL-2cells. The Bcl-2 and c-myc mRNA level remains unal-tered during this process (see Fig. 2), differentiating itfrom the cell death induced by interleukin-2 deprivationin these cell lines, when Bcl-2 and c-myc mRNA levelsare strongly reduced (Weller et al. 1994).

L1210 leukemic cells respond to TGF- with a partialarrest of the cell cycle at the G1/S transition and with theinduction of apoptotic cell death (Motyl et al. 1998). Theapoptotic process is accompanied by two phases of gen-eration of reactive oxygen species (ROS): a rapid(60 min) and a delayed (24 h and 48 h) phase after TGF- administration. Bcl-2 protein level decreases uponTGF- treatment, whereas the bax protein level increas-es, shifting the intracellular balance between death-pro-moting and death-inhibiting factors toward death induc-tion (Fig. 2).

The Ramos B-cell-lymphoma cell line also undergoesapoptosis in response to TGF- treatment. The apoptoticprocess in this cell line is accompanied by Bcl-X L down-regulation and the activation of caspase 3 (Saltzman etal. 1998). Downregulation of the anti-apoptotic Bcl-X Lprotein may, in this case, play a key role in cell-death in-

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Fig. 1 Schematic representation of the TGF- signal transductionpathway. Ligand binding to TGF- receptors leads to the forma-tion of heterotetrameric complexes of receptors I and II ( RI, RII ).TGF- RII phosphorylates and activates TGF- RI. Activated TGF-RI phosphorylates receptor-activated SMADs ( R-SMADs ), whichare released from a complex with SARA (SMAD anchor for recep-tor activation), a component important for targeting the R-SMADsto the receptor. The R-SMADs form a complex with a Co-SMADand enter the nucleus to interact with other co-activators or co-re-pressors for efficient transactivation or repression. Downregula-tion of the activated pathway is achieved in two ways. InhibitorySMADs ( I-SMAD ) are upregulated and prevent the activation of R-SMADs at the receptor level. After activation, R-SMADs aretargeted for degradation via the ubiquitin-proteasome pathway.Another TGF- signal, which is however not so well understood,is mediated via TAK1 (TGF- activated kinase 1). TAK1 may me-diate TGF- effects with respect to the activation of distinct MAP-kinase pathways ( JNK Jun N-terminal kinase, p38 stress activatedprotein kinase).


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duction, as the intracellular balance between pro- and an-ti-apoptotic molecules is disturbed.

Another mechanism of TGF- -induced cell death hasbeen proposed following the finding that TGF- down-regulates NF B activity by the induction of the expres-sion of I B, a specific inhibitor of NF B (Arsura et al.1996). I B binds to and inactivates NF B, a transcrip-tion factor that is involved in the mediation of survivalsignals. NF B inhibition results in the repression of c-myc expression in WEHI B cell lymphoma cells and in-creases cell death, which can be blocked by the ectopicexpression of c-myc (Arsura et al. 1996).

Several Eppstein Barr virus (EBV)-negative B-celllymphoma cell lines (BL-41, Ramos, and CAPA-2) re-spond to TGF- by the induction of apoptosis. The ex-pression of c-myc and Bcl-2 following TGF- treatmenthas been investigated and found to be unchanged. How-ever, another B-cell lymphoma cell line (CA46) and sev-eral preparations of normal human tonsilar B-cells do notundergo apoptotic cell death after TGF- stimulation.

These data suggest that there is a more complex regula-tory network of as yet uncharacterized intra- and/or ex-tracellular factors cooperating with TGF- to induce celldeath (Chaouchi et al. 1995).

One possibility is that immunosuppressants such ascyclosporine A can sensitize lymphocytes to Ca 2+-medi-ated cell death (Andjelic et al. 1997). Together withCa2+, cyclosporine induces the secretion of TGF- inB-cells. The synergistic action of TGF- and Ca 2+ subse-quently results in the apoptotic cell death of both T- andB-cells, thus leading to the speculation that TGF- mightplay a role in the cyclosporine A immunosuppressionpathway (Andjelic et al. 1997).

In another study, the upstream caspases involved inTGF- -mediated cell death have been studied in moredetail (Inman and Allday 2000). The TGF- -induced celldeath of the EBV-negative Burkitts lymphoma cell lineBL41 involves the activation of caspases 2, 3, 7, 8, and9. The authors demonstrate that the activation of the oth-er caspases is dependent on caspase 8 activation. How-

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Fig. 2 Schematic representa-tion of the apoptosis pathway.The apoptotic cascade can bedivided into three phases ( Initi-ation, Integration, Execution ).In the initiation phase, apopto-sis is triggered by stress signalsor specific factors actingthrough a subset of receptors.During the integration phase,

signals from several signalingpathways are balanced, and thedecision is made regardingwhether the execution of celldeath should be initiated. Dur-ing the execution phase, prote-ases of the caspase family areactivated and degrade specificsubstrates leading to the self-destruction of the cell. Severalkey steps and mediators areknown ( left ) for each phase.The events in which the TGF- pathway is involved are delin-eated right . The numbers indi-cate the respective references

in Table 1 (for a review of ap-optosis mechanisms, see Jarpeet al. 1998)


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ever, activation of caspase 8 in this cell system is inde-pendent of death-receptor activation by Fas or tumor ne-crosis factor (TNF- ). Bcl-X L and Bad protein levelswere reduced after 24 h, whereas the protein level of baxremained relatively unchanged.

The importance of TGF- for the maintenance of T-/ and B-cell homeostasis is best documented by in vivostudies. TGF- 1 knockout mice die 23 weeks after birthand show massive inflammation (Kulkarni et al. 1993;Shull et al. 1992). Transgenic mice overexpressing adominant negative mutant of the TGF RII, specificallyon T-cells, show a hyperplasia of CD8+ T-cells resultingin a massive expansion of lymph nodes and spleen(Lucas et al. 2000). However, TGF- 1 knockout micehave a smaller thymus and spleen than their wild-typelittermates (Shull et al. 1992; Christ et al. 1994), indica-tive of a depletion of T-cells, perhaps through inducedapoptosis. A recent publication from the group of SharonWahl (Chen et al. 2001) has expanded these findings bylocalizing TGF- 1 to mitochondria; this presumably me-diates a protective effect on T-cell apoptosis. These re-

sults are contradictory to other findings, and further in-vestigations are required into the possible ways in whichTGF- can mediate both pro- and anti-apoptotic stimuli.

The targeted deletion of TGF RII in B-cells has re-vealed the various roles of TGF- concerning the ho-meostasis and responsiveness of B-cell subpopulations(Cazac and Roes 2000). Cazac and Roes (2000) havefound that the B-cell response to a weak antigen shows aunusual hyper-responsiveness of B-cells manifested byextremely high IgG3 antibody production. Hyperplasiaof peripheral B-cells in the peritoneum and in Peyerspatches has been observed, revealing that the defect inB-cell homeostasis is the result of a defective cell-death

program.Taken together, there is thus strong evidence fromcell-culture experiments and in vivo data that TGF- is akey mediator of B- and T-cell homeostasis by regulatingproliferation processes and apoptosis in these cells.

TGF- -induced apoptosis in cells of the digestive system

As apoptotic cell death is a well-recognized phenomenonin the differentiation and maintenance of the liver, its un-derlying mechanism has been extensively studied in var-ious cell types originating in the liver. Rat liver epithelialcells undergo growth arrest and apoptosis upon TGF- treatment (Teramoto et al. 1998). Both p53 and bax areoverexpressed during the apoptotic process. Severalgrowth factors are unable to counteract this process,whereas interestingly, TGF- enhances TGF- mediatedapoptosis. TGF- alone also induces bax and p53 ex-pression leading to apoptosis without TGF- co-treat-ment. Thus TGF- and TGF- cooperate to induce apop-tosis in rat liver epithelial cells possibly via the inductionof p53 and bax. However, there are also reports of p53-independent pathways of TGF- -induced apoptosis(Yamamoto et al. 1996).

TGF- -induced cell death in Hep3B cells can be pre-vented by treatment with insulin (Chen and Chang 1997;Chuang et al. 1994) or by overexpression of IRS-1(Tanaka and Wands 1996). This insulin-mediated survivalsignal acts via activation of PI 3-kinase and Akt. Consti-tutively active Akt can mimic the anti-apoptotic effect of insulin, and the anti-apoptotic effect of insulin can be in-hibited by a dominant negative Akt. This kinase pathway

seems to influence the activation of caspase 3 down-stream of activated SMADs, because SMAD activationand translocation into the nucleus remains unaffected bythe PI 3-kinase/Akt pathway (Chen et al. 1998).

Analyses of TGF- induced cell death in the Hep3Bhepatoma cell line have confirmed that apoptosis is ac-companied by the activation of caspase-family proteases(Hung et al. 1998). The broad-spectrum caspase inhibitorZVAD-FMK blocks TGF- -induced cell death in a con-centration-dependent manner. Application of the morespecific caspase inhibitor DEVD-FMK has revealed thatthe activation of caspase 3 is necessary for TGF- -in-duced cell death. Immunoblotting has confirmed the

cleavage of the caspase-3-specific substrate poly(ADP-ribose)polymerase (PARP) after TGF- treatment (Hunget al. 1998).

Interestingly, McGinnis et al. (1999) have shown thatPARP and procaspase 3 are substrates of calpain in vitroand in vivo. This finding suggests that TGF- -mediatedapoptosis may not depend on the SMAD pathway, butmay involve another mechanism directly activating cal-pain and subsequently the caspase cascade. In this con-text, Gressner and coworkers (1997) have been able toshow that TGF- -induced cell death in primary rat hepa-tocytes can be inhibited by the administration of calpaininhibitors.

Analysis of the apoptotic process in isolated primaryhepatocytes of the rat has revealed that transcriptionalactivation is necessary for cell-death induction (Inayat-Hussain et al. 1997). The application of cycloheximide,which blocks de novo protein sythesis, can block TGF--mediated apoptosis induction. Caspase 3 is activated,whereas caspase 1 is not increased in this process.

Shima and coworkers (1999) have detected the activa-tion of caspase 8, 9, and 3 in response to TGF- admin-istration to HuH-7 hepatoma cells. The apoptotic processis accompanied by a decreased level of Bcl-X L proteinand a low Bax/Bcl-X L ratio. TGF- treatment results in areduced level of XIAP, an inhibitor of apoptosis previ-ously shown to interact with the TAK1/TAB1 complex(TGF- -activated kinase/TAK-binding protein), down-modulating the kinase activity of TAK1 (Shibuya et al.1996). Interestingly, the potent growth factor, epidermalgrowth factor (EGF), completely abolishes the TGF- -induced apoptosis of HuH-7 cells by an unknown mech-anism, as XIAP and Bcl-X L levels remain unaffected(Shima et al. 1999).

The investigation of human (HepG2), mouse(55.1c), and rat (FTO-2B) hepatoma cell lines has re-vealed differences in their response to TGF- (Buene-mann et al. 2001). The activation of the SMAD path-

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way, monitored by measurement of luciferase activityfrom a SMAD-driven promoter element, is similar inall three cell lines, in which transient expression of TGF- induced early gene (TIEG) is observed. Where-as HepG2 cells are completely resistant to TGF- -in-duced apoptosis and growth arrest, 55.1c cells show on-ly modest apoptosis. FTO-2B cells undergo G1 arrestand massive apoptosis in response to TGF- . Since theSMAD pathway and TIEG expression, which are in-volved in TGF- -mediated signaling and the inductionof cell death, are similar in all three cell lines, the de-fect in the cell-death pathway in HepG2 and 55.1c mustlie downstream of SMAD and TIEG (Buenemann et al.2001).

The role of TIEG in apoptosis induction has been in-vestigated in Hep 3B cells (Ribeiro et al. 1999). LikeTGF- , TIEG induces the generation of ROS and theloss of the mitochondrial membrane potential precedingthe apoptotic death of TIEG-transfected cells.

In the rat hepatoma cell line FaO, TGF- induces ap-optosis via the activation of caspases 2, 3, 7, and 8

(Freathy et al. 2000). The activation of caspase 3 and 7 isblocked by a broad-spectrum caspase inhibitor; however,the assembly of the apoptosome complex is not dis-turbed. The process of cytochrome C release and apopto-some assembly triggered by TGF- is not accompaniedby the downregulation of Bcl-XL protein, an event oftendescribed for other cell systems. Freathy et al. (2000)speculate that another Bcl-2-family-independent mecha-nism, such as the generation of ROS, may be involved.However, they do not address this question further byconclusive experiments.

In primary hepatocytes, TNF- and EGF antagonizethe pro-apoptotic effect of TGF- (Roberts et al. 2000).

Pretreatment of hepatocytes with EGF or TNF- sup-presses TGF- -induced cell death by 73% and 50%, re-spectively. Various survival signals are involved in thisprocess. Whereas EGF relays signals via phosphoinosi-tide 3-kinase (PI 3-kinase) and extracellular signal regu-lated kinase (ERK), the suppression of TGF- -inducedapoptosis by TNF- is mediated by ERK and p38 MAPkinase. Again, this study provides strong evidence thatTGF- signaling interacts with other signaling pathwaysto regulate cellular survival.

In fetal hepatocytes, TGF- induces growth arrest inthe G1 phase of the cell cycle (Sanchez et al. 1995).Moreover, TGF- induces apoptosis of fetal hepatocytesvia a mechanism that requires new protein synthesis(Sanchez et al. 1997). Together with EGF, TGF- pro-motes the differentiation of these cells (Sanchez et al.1998). In the context of these studies, it has been ob-served that, regardless of the concentration of TGF- ,50% of the cells always survive. These cells are less dif-ferentiated with respect to liver-specific transcriptionfactor activity and are still able to undergo growth arrestin response to TGF- , whereas they are totally resistantto TGF- -mediated apoptosis (Sanchez et al. 1999).These data suggest a role for TGF- during liver devel-opment and differentiation.

TGF- -induced cell death in the reproductive system

Apoptosis occurs in specific uterine cells during the es-trous cycle, blastocyst implantation, and decidualization.Active proliferation of endometrial stromal cells duringthe decidualization of early pregnancy are followed byprogrammed cell death in the antimesometrial region of the decidua. The control of the proliferation and pattern

formation of cellular structures in the placenta after blas-tocyst implantation depends on the synthesis and secre-tion of endometrial growth factors (Moulton 1994). ThemRNAs for TGF- 1 and TGF- 2 have been detected indecidual cells on day 6.5 of pregnancy. To investigatethe role of TGF- in the apoptosis of decidual cells,Moulton (1994) isolated endometrial stromal cells fromprogestin-pretreated ovariectomized rats. Application of exogenous TGF 1 to the cultured cells induced celldeath in a concentration-dependent manner. TGF- 2showed the same effect. Speculating that a paracrine/au-tocrine mechanism of cell-death induction was responsi-ble for the observed cell death in vivo, Moulton (1994)

tested the hypothesis with high density cultures of endo-metrial cells, assuming that a high concentration of se-creted TGF- would increase cell death. With antibodiesagainst TGF- 1 and - 2, only the active form of TGF- 2could be detected within the cell culture supernatant, in-dicating that TGF- 2 was responsible for the TGF- -me-diated effects in vivo.

TGF- has been shown to induce apoptosis in normalprostate cells and prostatic carcinoma cells in vitro andin vivo (Hsing et al. 1996; Kyprianou and Isaacs 1989;Landstrm et al. 1996). Castration of Dunning R3327PAP rats leads to the upregulation of TGF- 1 and bothTGF- receptors, correlating with increased apoptosis

(Landstrm et al. 1996). SMAD proteins are involved inthis process as revealed by another study showing en-hanced SMAD expression and activated SMAD2 and nu-clear SMAD6 and SMAD7 in areas with a large numberof apoptotic cells (Brodin et al. 1999).

One example of in vitro studies with prostate cancercells is the TGF- -induced apoptosis in PC-3 cells(Rajah et al. 1997). The apoptotic process here is accom-panied by an increase in the expression of insulin-likegrowth-factor-binding protein 3 (IGFBP-3), another in-hibitor of cell growth. IGFBP-3 is thought to act via thesequestration of free IGFs and, thereby, the hindrance of the activation of IGF receptors. Rajah et al. (1997) couldinduce apoptosis by IGFBP-3 treatment of the cells. Fur-thermore, they could inhibit TGF- -mediated apoptosiswith neutralizing antibodies against IGFBP-3. In this cellline, TGF- -induced cell death may be mediated viaIGFBP-3 (Rajah et al. 1997). In a study by Hsing and co-workers (1996), TGF- -induced cell death has beenanalysed in nontumorigenic NRP-152 cells and tumori-genic NRP-154 prostatic epithelial cells. In this cellmodel, dexamethasone (Dex) enhances TGF- -mediatedapoptosis, whereas IGF-I counteracts apoptosis induc-tion. This may support the data of Rajah and coworkers(1997) who have shown that TGF- -mediated cell death

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is induced via the upregulation of IGFBP-3, which se-questers IGF-I and hinders the survival promoting effectof this growth factor.

Apoptosis is an important process during the remod-eling of the breast after postlactational involution. TGF- mRNA is expressed at low levels in the mammarygland of pregnant mice but increases dramatically withinthe first 2 days following weaning and continues to be

elevated throughout postlactational involution (Rosfjordand Dickson 1999). The high expression of TGF- intransgenic mice results in the lack of secretory lobule de-velopment and an inability to lactate accompanied by in-creased apoptotic cell death within the mammary gland.These data indicate that TGF- is important for tissue re-modeling in vivo, possibly by the regulation of apoptoticcell death during distinct phases during the animal lifecycle (Rosfjord and Dickson 1999).

Additional evidence from in vitro studies supports therole of TGF- as a death-inducing factor in the mamma-ry gland. TGF- induces apoptosis in c-myc overex-pressing mouse mammary epithelial cells (Amundadottir

et al. 1996; Nass et al. 1996). Cell death is accompaniedby a substantial downregulation of Bcl-2 and Bcl-X LmRNA. TGF- can also directly induce cell death inMCF-7 breast cancer cells (Chen et al. 1996).

Perry and coworkers (1995) have shown that TGF- is induced by tamoxifen treatment in MCF-7 and MBA-MD-231 breast cancer cell lines. Tamoxifen-induced celldeath can be prevented by TGF- neutralizing antibod-ies.

TGF- also induces cell death in NIH-OVCAR-3ovarian carcinoma cells. As in other TGF- -treated cells,the Bcl-2 protein level is decreased (Lafon et al. 1996).Here, the apoptotic process can be inhibited by the ad-

ministration of N-acetylcysteine, an antioxidative agent,thus revealing an important role for ROS in this cell line.

TGF- -induced cell death in tissue formationand remodeling

TGF- induces capillary morphogenesis in vitro and an-giogenesis in vivo. Using a dominant negative TGF-RII, Choi and Ballermann (1995) have shown that ex-ogenous and endogenous TGF- induces growth arrestand capillary formation accompanied by massive celldeath during this morphogenetic process. This TGF- ef-fect is mediated via receptor activation, because cellstransfected with the mutant receptor do not respond toTGF- . The application of TGF- neutralizing antibod-ies has substantiated this finding, indicating that TGF- -mediated apoptosis may be of great relevance during em-bryogenetic tissue modeling (Choi and Ballermann1995).

In human aortic smooth muscle cells, TGF- treat-ment results in massive apoptotic cell death (Hishikawaet al. 1999). Since a TGF- -responsive element existsin the connective tissue growth factor (CTGF) gene,Hishikawa et al. (1999) investigated the role of this fac-

tor in TGF- -mediated apoptosis. They found that CTGFwas upregulated by TGF- . Inhibition of CTGF by anti-sense oligonucleotides inhibited TGF- -induced celldeath and reduced caspase 3 activation by TGF- . Thesedata suggest an important role of CTGF in TGF- -medi-ated cell death in human aortic smooth muscle cells pos-sibly via autocrine mechanisms (Hishikawa et al. 1999).

Apoptosis is believed to be important for proper

wound healing. During the maturation of the wound, ex-cess cell numbers are reduced by programmed cell death(Crowe et al. 2000). During wound healing, apoptoticcells are mainly localized in the granulation tissue be-neath the advancing epithelial edge. TGF- 1/Scid dou-ble-knockout mice reveal a delay in each of the majorphases of wound healing, including a delayed and re-duced onset of apoptosis in cells localized in the granula-tion tissue (Crowe et al. 2000). Thus, the TGF- s havebeen implicated in wound healing, possibly via their ap-optosis-inducing capacity. The data also suggest a com-pensatory role for TGF- 2 and 3, as these are upregulat-ed in TGF- 1/Scid knockout mice and may be responsi-

ble for succesful healing of the wound.TGF- 3 knockout mice exhibit, among other distor-tions, a cleft palate (Proetzel et al. 1995). Detailed analy-sis of this phenotype has revealed that medial edge epi-thelial cells undergo apoptosis during the process of cleftfusion (Martinez-Alvarez et al. 2000). In palates of TGF-3 knockout mice, apoptotic cell death is markedly re-duced. This, together with other observations in theseknockout animals, provides good evidence that the apop-tosis-inducing capacity of TGF- 3 is necessary, at leastin part, for successful palatal fusion.

TGF- -induced cell death in the nervous systemThe regulation of the balance of neuron survival anddeath is a permanent feature in nervous system develop-ment, maintenance, degeneration, and repair. The firstevidence for a role of TGF- in the regulation of apopto-sis in the nervous system came from the following in vit-ro study. Experiments by de Luca and coworkers (1996)showed that cultured immature cerebellar granule neu-rons died apoptotically within several days in vitro un-less they were cultured with high concentrations of po-tassium, which led to depolarization. All three TGF- isoforms induced apoptosis when these neurons weremaintained under low potassium levels. This effect couldnot be blocked by the application of CNTF or LIF, cyto-kines that enhance neuronal survival, or IGF-I, whichnormally prevents apoptosis after potassium withdrawal.Interestingly, such neurons cultured for several days inhigh potassium acquired resistance to TGF- -mediatedcell death. These findings imply that TGF- limits theexpansion of postmitotic neuronal precursor populations,on the one hand, by promoting cell death and, on the oth-er hand, by supporting the survival of those neurons thathave succesfully reached their target area and gainedsupportive synaptic connectivity (de Luca et al. 1996).

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The role of TGF- in ontogenetic neuron death in vivowas first demonstrated in a study in which chick embryoswere used as a model system (Krieglstein et al. 2000). Byapplication of TGF- neutralizing antibodies in ovo, theontogenetic death of ciliary, dorsal root, and spinal motorneurons was largely prevented. Moreover, in limb-budablation experiments, the classical protocol used to showthe importance of target-derived survival factors (Holly-

day and Hamburger 1976; Oppenheim 1991; Pettmannand Henderson 1998) during the phase of ontogeneticneuron death, the immunoneutralization of TGF- res-cued both motoneurons and dorsal root ganglion neuronsthat would normally die (Krieglstein et al. 2000).

In another study, it could be shown that TGF- is es-sentially required to regulate programmed cell death inthe central retina (Dnker et al. 2001). Application of TGF- neutralizing antibodies resulted in a substantialdecrease of TUNEL-positive cells in the central retina.This decrease was comparable with the effect of nervegrowth factor (NGF) immunodepletion in chick embryo-nal retina (Dnker et al. 2001; Frade et al. 1996).

Furthermore, in primary rat oligodendrocyte progeni-tor cultures, TGF- induces both growth arrest and apop-tosis (Yu et al. 2000). TGF- -induced cell death can beprevented when the cells are pretreated for 15 h withFGF-2, whereas TNF- -induced cell death is not affect-ed. Yu et al. (2000) have unfortunately not investigatedthe apoptosis-inducing cascade, their experiments beingdirected mainly at the mechanisms involved in the G1arrest.

TGF- inhibits cell proliferation of T24 glioma and47616 trigeminal neurinoma cells. Inhibition of cellgrowth is accompanied by apoptotic cell death (Marushigeand Marushige 1994). The mode of cell death between

these two cell lines seems to be different. Cells of the47616 line undergo apoptosis primarily after growth ar-rest in G1. Apoptosis can be stimulated by serum with-drawal and inhibited by mitogenic stimuli, such as insu-lin and platelet-derived growth factor. T24 cells undergoonly a moderate growth arrest after TGF- treatment anddie by apoptosis when cells reach confluency in the cul-ture dish. The kinase inhibitor staurosporine can acceler-ate TGF- -induced apoptosis in both 47616 and T24cells, whereas phorbol ester treatment inhibits apoptoticcell death in 47616 cells but stimulates that of T24cells. These results clearly indicate that there are at leasttwo different modes of TGF- -induced apoptosis withincultured cells (Marushige and Marushige 1994).

In a study by de Luca et al. (1996), the authors inves-tigated the role of TGF- expression in the progressionof malignant glioma by using an immunocompetent mu-rine model. They generated cell lines stably overexpress-ing TGF- or repressing endogenous expression of TGF-. These cells were injected either subcutaneously or in-tracranially. TGF- inhibited the induction of anti-tumorcytotoxicity when the tumor cells were applied subcuta-neously but not when they were injected intracranially.Overproduction of TGF- reduced the tumorigenicity of tumors induced by subcutaneous or intracranial injection

in line with an increase of apoptosis within the tumor ar-ea. The opposite effect was observed with the antisensecell lines, which underproduced TGF- . This resulted ina higher growth rate of the tumor and reduced apoptoticcell death within the tumor area. Furthermore, de Luca etal. (1996) could show that Fas/APO-1 was expressed invitro and in vivo in the parental SMA 560 cell line. Invitro experiments showed that these cell line was sensi-

tive to Fas- and TGF- -induced cell death. Applicationof both factors synergistically potentiated apoptotic celldeath (Ashley et al. 1998).

Synergistic effects

The cooperativity of TGF- with other growth factors isone possible explanation for its multifunctional proper-ties. Within the last few years, data have accumulatedsuggesting that TGF- modulates the action of othergrowth factors, i.e., the neurotrophic function of GDNF.TGF- synergizes with GDNF to promote the survival of

peripheral and central nervous system dopaminergic neu-rons (Krieglstein et al. 1998; Unsicker and Krieglstein2000). On the other hand, TGF- and TNF- act syner-gistically to induce cell death in Schwann cells (Skoff etal. 1998). Neither TNF- nor TGF- alone are capableof inducing cell death in these cells, despite these growthfactors individually inducing cell death in a variety of different cell types.

TGF- enhances growth inhibitory effects of Dex and1,25-dihydroxyvitamin D 3 (VD3) in monocytoid leuke-mia U937 cells (Kanatani et al. 1999). The expression of differentiation specific markers, such as CD11b andCD14 antigens, is enhanced by VD3. In cooperation with

Dex, these genes are not induced, but the number of apo-ptotic cells (Apo2.7-positive cells) is increased. Bothfactors induce, together with TGF- , an increased ex-pression of p21 waf1 leading to a strong hypophosphoryla-tion of the retinoblastoma-susceptibility gene productpRb. When cells are treated with TGF- and Dex incombination, the anti-apoptotic Bcl-X L protein is down-regulated, whereas co-treatment with VD3 blocks thedownregulation of Bcl-X L. These data suggest that TGF- is an indispensable cofactor either for differentiation pro-cesses or for the elimination of cells, depending on thecooperating growth factor (Kanatani et al. 1999).

In thecal/interstitial cells, TGF- and TGF- syner-gistically induce apoptosis, whereas each growth factorby itself has no effect on cell viability. In T/I cells treatedwith both growth factors, Bcl-2 mRNA levels decreasesignificantly, and the expression of ICE/caspase-1 is en-hanced three-fold. Cells treated only with TGF- orTGF- show unaltered Bcl-2 and caspase-1 mRNA lev-els. Induction and repression processes are involved inthe synergistic action of TGF- and TGF- . This per-haps could explain why only both growth factors togeth-er can induce cell death in these cells, one componentbeing responsible for induction, the other for repression(Foghi et al. 1998).

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TGF- induces cell death in transformed fibroblastsand synergizes with TNF- in this process (Schulz andBauer 2000). Pretreatment of cells with TNF- followedby application of TGF- causes a faster induction of ap-optosis and vice versa. The underlying mechanism seemsto be the reduction in the level of endogenous survivalfactors.

The results from an interesting study conducted on

MCF-7 cells (Tobin et al. 2001) lend support for the pu-tative role of the TGF- pathway in TNF- -induced ap-optosis. A dominant negative TGF- receptor was in-duced to be overexpressed in order to disturb TGF- sig-naling in the MCF-7 cells. These MCF-7 cells were rela-tively resistant to TNF- -induced cell death, a resistancepossibly mediated by the increased Bcl-2 expression inthese cells. Levels of bax and Bcl-X L remained un-changed (Tobin et al. 2001). These data suggest thatTGF- sensitizes cells for other pro-apoptotic stimuli bythe downregulation of anti-apoptotic Bcl-2.

Another study has demonstrated a synergism betweenEGF and TGF- in the induction of apoptosis in porcine

thyroid follicles (Bechtner et al. 1999). However, thisdoes not seem to be real synergism as EGF induces theexpression of TGF- in this cell system, and therfore, theincreased TGF- level could account for the increase inapoptotic cells. Nevertheless, the induction of cell deathby TGF- is dose-dependent. The application of IGFcould not rescue the cells from TGF- -induced cell death(Bechtner et al. 1999).

Cell death induced by TGF- effectors

TGF- sends signals to the nucleus via SMAD-family

member proteins (Massague 2000). The overexpressionof DPC4 (also called SMAD4) results in the induction of apoptosis in transiently transfected MDCK cells (Atfi etal. 1997). Coexpression of SMAD3 and SMAD4 resultsin an enhancement of apoptosis. SMAD4 and SMAD3together could enhance TGF- -mediated transactivation.Atfi et al. (1997) have shown that supertransactivation of TGF- -responsive genes by SMAD3/SMAD4 coexpres-sion can be blocked by the dominant negative c-jun(TAM67). Similar effects have been obtained with thedominant negative MEKK1, a kinase involved in the ac-tivation of the JNK-MAP kinase pathway. These find-ings suggest that TGF- signaling processes are depen-dent on other signal pathways for an optimal outcomeand that ectopic expression of SMAD proteins can in-duce apoptosis (Atfi et al. 1997).

SMAD2 and SMAD3 are highly homologous mem-bers of the receptor-activated SMADs. Yanagisawa andcoworkers (1998) found that, upon overexpression in sta-bly transfected cell lines, cell death was strongly inducedby SMAD3, but to a much lesser extend by SMAD2, inthe presence of TGF- . Interestingly, human lung epithe-lial cells downregulated SMAD3 expression after TGF- administration, whereas SMAD2 expression remainedunaffected. These data demonstrated, for the first time,

the functional differences between these two TGF- ef-fector molecules in vivo (Yanagisawa et al. 1998).

The importance of the SMAD pathway for TGF- -mediated apoptosis has been substantiated by a study of Yamamura and coworkers (2000). They investigatedcell-death induction in M1 and Hep3B cells and foundthat the overexpression of dominant negative SMAD3inhibited TGF- -induced apoptosis (Yamamura et al.2000). The same effect could be achieved by the overex-pression of the inhibitory SMAD7. More interestingly,an interaction between SMAD proteins and the AP1transcription factor was found to be involved. The inhi-bition of AP1 by the overexpression of a dominant nega-tive FosB also caused substantial inhibition of TGF- -mediated apoptosis. Moreover, the overexpression of JunD-FosB potentiated TGF- -mediated cell death, cor-roborating the importance of other signaling pathwaysfor TGF- -mediated apoptosis.

Although SMAD7 has been described as an inhibitorySMAD protein that prevents the phosphorylation of re-ceptor-activated SMADs, thereby inhibiting their nuclear

translocation and the TGF- -induced response (ten Dijkeet al. 2000), SMAD7 has been shown to possess the ca-pacity to induce apoptosis in prostatic carcinoma cells(Landstrm et al. 2000). Ectopic expression of SMAD7induces apoptosis in PC-3U cells. The expression of SMAD7 antisense mRNA abolishes TGF- -induced celldeath in these cells. The same effect can be observed inTGF- -treated DU 145 and HaCaT cells when SMAD7antisense mRNA is expressed. Taken together, these re-sults provide evidence for a new effector role of SMAD7during TGF- -induced apoptosis (Landstrm et al.2000). Although this seems to be contradictory to the re-sults obtained by Patil and coworkers (2000) who have

described an anti-apoptotive effect in B-cells, the viewof SMAD7 as an apoptosis-inducing protein is substanti-ated by the findings of Lallemand et al. (2001). SMAD7seems to induce the JNK-signaling cascade specifically,whereas p38 and p42/p44 MAPK are not affected. Theactivation of the JNK pathway however is not involvedin the inhibitory function of SMAD7 that prevents theactivation of SMAD2/3 at the receptor level, whereas itis necessary for SMAD7-induced apoptosis (Lallemandet al. 2001).

The adapter protein Daxx seems to be another essen-tial component of the TGF- cell-death pathway(Perlman et al. 2001). Daxx, which is normally associ-ated with the Fas receptor and mediates the activationof JNK and Fas-induced apoptosis, has been found tobind directly to the cytoplasmic domain of TGF RII.Treatment of AML12 mouse hepatocytes with antisenseoligonucleotides for Daxx results in substantial inhibi-tion of TGF- -mediated apoptosis. The induction of ap-optosis and the activation of JNK by TGF- applicationcan be abolished by the overexpression of the dominantnegative c-terminal part of the Daxx protein, indicatingthat JNK activation via Daxx is an important upstreamevent during TGF- -mediated apoptosis (Perlman et al.2001).

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10

Table 1 (continued)

No. Cell type TGF- Genes/proteins Activated Other factors Co- Referenceiso- upregulated ( )/ caspase operationform downregulated ( )/

unchanged ()/ other modifications

24 Ramos - 1 bcl-X L , bik 3 Saltzman et al.1998

25 WEHI - 1 I B , c-myc ND Ectopic expression of c-myc Arsura et al.inhibits apoptosis 1996

26 B- and T-cells - 1 ND ND Ca 2+ and cyclosporin induce Ca 2+ Andjelic et al.TGF- expression cyclosporin 1997

27 Schwann cells - 1 ND ND ND TNF- Skoff et al. 199828 Primary - 1 p21 waf1 , p27 kip1 ND Yu et al. 2000

oligodendrocytes29 SMA 560 glioma - 1 ND ND Fas Ashley et al.

cells 199830 Glioma and - 1 ND ND Inhibition of apoptosis by Marushige and

trigeminal insulin and platelet-derived Marushige 1994neurinoma cells growth factor

31 Cerebellar granule - 1, ND ND Apoptosis under low de Luca et al.neurons - 2, potassium concentration; 1996

-3 no reversion of apoptosisby CNTF, LIF, and IGF-I32 In vivo; neutral- ND 3 Cell death of DRG, CG, Krieglstein et al.

izing antibodies; and motoneurons is reduced 2000chick embryo after TGF- neutralization

33 In vivo; neutral- ND ND Developmental cell death NGF Dnker et al.izing antibodies; in the retina is reduced 2001chick embryo after TGF- neutralization

34 Transformed - 1 Levels of anti-apoptotic Synergism with TNF- TNF- Schulz and Bauerfibroblasts proteins are reduced 2000

35 Capillary - 1 ND ND Dominant negative Choi andendothelial cells TGF- RII blocks apoptosis Ballermann 1995

36 Aortic smooth - 1 CTGF 3 TGF- effect is mediated Hishikawa et al.muscle cells by CTGF 1999

37 Lung epithelial SMAD3 ND SMAD3 induces apoptosis Yanagisawa et al.cells 199838 Thecal interstitial bcl-2 mRNA , 1 TGF- Foghi et al. 1998

cells caspase1 expression 39 Thyroid follicle - 1 ND ND EGF stimulates EGF Bechtner et al.

TGF- 1 expression 199940 NIH-OVCAR-3 - 1 fos , jun , bcl-2 , ROS induction; Lafon et al. 1996

ovarian carcinoma N-acetylcysteine and ectopiccells bcl-2 expression inhibits

apoptosis41 MCF-7, ND ND Perry et al. 1995

MBA-MD-231breast cancer cells

42 MCF-7 - 1 bax (), bcl-2 , ND Dominant negative TNF- Tobin et al. 2001bcl-X L () in DNRII TGF- RII reducesexpressing cells TNF- mediated apoptosis

43 Mammary - 1 bcl-X L ND EGF counteracts TGF- Nass et al. 1996epithelial cells

44 Mammary gland ND ND Remodeling of gland tissue Rosfjord andafter weaning involves Dickson 1999apoptosis modulated byTGF- ; IGF-I and TGF- inhibits remodeling andapoptosis

45 Endometrial - 1, -2 ND ND Moulton 1994stromal cells


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46 In vivo, prostate SMAD3, 4 , ND Castration paradigm Brodin et al.Dunning R3327 activated SMAD2 , 1999rat model SMAD6 , SMAD7 ,nuclear localized

SMAD747 Prostatic - 1 SMAD7 ND SMAD7 induces apoptosis, Landstrm et al.

carcinoma cells antisense SMAD7 2000inhibits apoptosis

48 PC-3 - 1 IGFBP-3 Yes, not IGFBP-3 induces apoptosis Rajah et al. 1997specified

49 NRP-152/ - 1, ND ND Dex enhances apoptosis; Dex Hsing et al. 1996NRP-154 Prostate - 2, IGF-I inhibits apoptosisepithelial cells - 3

50 In vivo; - 1 ND ND TGF- 2, and 3 compensate Crowe et al. 2000TGF- 1-Scid 1 loss; apoptosis duringdouble knockout wound healing is disturbed

51 In vivo; - 3 ND ND Medial edge epithelial cell Martinez-AlvarezTGF- 3 apoptosis is disturbed and et al. 2000knockout responsible for palate

fusion defect52 MDCK cells JNK activation ND SMAD4 expression induces Atfi et al. 1997

apoptosis; JNK activation;dominant negative jun caninhibit apoptosis

53 Mv1Lu, JNK activation ND SMAD7 induces apoptosis; Mazars et al.COS-7, HepG2 SMAD7-induced inhibition 2001

of receptor signaling isindependent of JNK activation

54 Pancreatic TIEG induces apoptosis Tachibana et al.epithelial cells 1997

55 NRP-1, COS-1, - 1 3 ARTS sensitizes cells for Larisch et al.MCF-7 cells TGF- mediated apoptosis: 2000

upon TGF- stimulation,ARTS translocatesfrom mitochondriato the nucleus

56 COS-1, - 1 JNK activation ND Daxx adapter protein is Perlman et al.AML12 cells reqired for TGF- induced 2001

apoptosis and JNK activation

11

Table 1 (continued)

No. Cell type TGF- Genes/proteins Activated Other factors Co- Referenceiso- upregulated ( )/ caspase operationform downregulated ( )/

unchanged ()/ other modifications

Another downstream effector of TGF- function isthe SP1-like zinc-finger protein TIEG, which was firstidentified in human osteoblasts (Subramaniam et al.1995). Ectopic overexpression of TIEG has been shownto induce apoptosis in epithelial cells, hepatocytes, andpancreatic epithelial cells (Chalaux et al. 1999; Ribeiroet al. 1999; Tachibana et al. 1997). TIEG action seems toinvolve the activation of ROS, as the blocking of ROSgeneration could block TIEG-mediated apoptosis (Ribeiroet al. 1999).

Recently, the group of Anita Roberts has describedthe finding of the new protein ARTS (apoptosis-relatedprotein in the TGF- signaling pathway), a new TGF- downstream effector important for TGF- -mediated ap-optosis (Larisch et al. 2000). ARTS is a 32-kDa septin-like protein that has been located in mitochondria. ARTSsensitizes cells to TGF- -stimulated apoptosis and af-

fects only weakly fas or TNF- -mediated apoptosis. Up-on TGF- stimulation, ARTS loses its mitochondrial lo-calization and migrates to the nucleus. The inhibition of ARTS protein expression by antisense technology inhib-its TGF- -stimulated apoptosis substantially, showingthe important role of ARTS during this process (Larischet al. 2000).

Conclusions

Taken all the available data regarding TGF- and TGF--mediated apoptosis together, it is clear that TGF- it-self is a death-inducing agent in vitro and in vivo (Ta-ble 1). However, it is also clear that the death-inducingcapacity of TGF- is context-dependent, i.e., it is re-stricted to certain cell types, to a certain state of differen-


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tiation, and most notably, to the presence or absence of other growth factors. This may explain the contradictoryresults of some studies, e.g., when considering the roleof SMAD7. Remaining unresolved issues include theway in which caspase activation is regulated by TGF- or whether the downregulation of the anti-apoptotic pro-teins Bcl-2 and Bcl-X L is a critical step during TGF- -mediated apoptosis. Furthermore, a great deal of work

remains concerning the regulatory molecules and theirimportance for TGF- -induced apoptosis, in order togenerate a complete and conclusive model that also ex-plains the many interactions with other signaling path-ways.

Acknowledgements The authors would like to thank HerdisBender and Dr. Ziyuan Wang for critical reading of the manu-script.

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