(Journal of Leukocyte Biology. 2002;71:731-740.)
© 2002
by Society for Leukocyte Biology
Transforming growth factor ß signal transduction
Sylviane Dennler,
Marie-José Goumans and
Peter ten Dijke
Division of Cellular Biochemistry, The Netherlands Cancer Institute, Amsterdam, The Netherlands
Correspondence: Peter ten Dijke, Division of Cellular Biochemistry (H3), The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. E-mail: p.t.dijke{at}nki.nl
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ABSTRACT
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Transforming growth factor beta1 (TGF-ß1) is the prototypic member of a large family of structurally related pleiotropic-secreted cytokines that play a pivotal role in the control of differentiation, proliferation, and state of activation of many different cell types including immune cells. TGF-ß family members have potent immunosuppressor activities in vitro and in vivo. These cytokines trigger their biological effects by inducing the formation of a heteromeric transmembrane serine/threonine kinase receptor complex. These receptors then initiate intracellular signaling through activation of Smad proteins, and specific Smads become phosphorylated and associate with other Smads. These heteromeric Smad complexes accumulate in the nucleus, where they modulate the expression of target genes. Recent data support the notion that Smads are important intracellular effectors of TGF-ß in immune cells. Here, we review recent advances in TGF-ß signal transduction in immune cells.
Key Words: immune system • Smad • transcription factor
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INTRODUCTION
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The transforming growth factor beta (TGF-ß) superfamily consists of more than 30 related members in mammals, including 3 TGF-ßs, 4 activins, and over 20 bone morphogenetic proteins (BMPs) [1
, 2
]. TGF-ß was named after its ability to induce the growth of normal rat kidney cells in soft agar [3
]. Subsequently, TGF-ß was shown to be a potent growth inhibitor [4
] and found to be a multifunctional protein (Fig. 1
) [5
]. TGF-ß modulates cell proliferation, differentiation, apoptosis, adhesion, and migration of various cell types and favors the production of extracellular matrix proteins. Most cell types, including immature hematopoietic cells, activated T and B cells, macrophages, neutrophils, and dendritic cells, produce TGF-ß and/or are sensitive to its effects [6
7
8
]. Activin was identified originally as a factor that stimulates the secretion of follicle-stimulating hormone from the pituitary gland [9
] and as a stimulator of erythroid differentiation [10
]. BMP was first known for its ability to induce bone and cartilage formation [11
]. Subsequent studies about activin and BMP have revealed that these cytokines, such as TGF-ß, have a broad range of activities and act on many different cell types. BMP and activin are likely to have important effects on immune cells. Loss of function studies with TGF-ß family ligands in mice has also demonstrated their multifunctional properties and revealed their important role during embryogenesis and in maintaining homeostasis during adult life [12
]. Subversion of TGF-ß family signaling has been implicated in various human diseases, including autoimmune diseases, vascular disorders, and cancer (Fig. 1)
[13
].
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Figure 1. TGF-ß is a multifunctional regulator. For example, TGF-ß inhibits cell growth and promotes apoptosis, stimulates extracellular matrix production, regulates the proliferation and migration of endothelial and vascular smooth muscle cells, and regulates the proliferation, differentiation, and activation of immune cells. Deregulated TGF-ß signaling has been implicated in various human diseases, including cancer, fibrosis, vascular disorders, and autoimmune diseases.
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The pleiotropic action of TGF-ß family members implicates the need for tight regulation of their biological activities. Consistent with this notion, TGF-ß is secreted predominantly as a latent complex that has to be activated before being capable of eliciting biological effects [14
]. TGF-ß is synthesized as a precursor that is proteolytically processed; the N-terminal domain, also termed latency-associated peptide, remains noncovalently associated with the C-terminal bioactive dimer and prevents the binding of TGF-ß to its specific cell-surface receptor. The mature TGF-ß protein can be released from the inactive complex by action of proteases, such as plasmin, or through the action of binding proteins, such as thrombospondin [15
]. Latent complexes for activins and BMPs have not been described. However, the biological activities of activins and BMPs are also regulated tightly through their interaction with many specific extracellular inhibitors, such as follistatin and noggin [16
, 17
].
Another property consistent with the multifunctional role of TGF-ß family ligands is that they usually act in an autocrine or paracrine manner and that the biological half-life of TGF-ß family members is short [18
]. TGF-ß family ligands are rapidly cleared through the action of scavenger molecules, such as 
2-macroglobulin [19
, 20
]. However, it appears that TGF-ß family members may also have an endocrine mode of action. Evidence for a physiological role of circulating TGF-ß1 was revealed by the analysis of TGF-ß1-deficient mice [21
, 22
]. Depending on the genetic background, half of these mice die in utero, whereas the other half makes it to term. The embryonic rescue of the pups can be partially explained by the TGF-ß1, which is transferred transplacentally and lactationally from the heterozygous mothers to their TGF-ß1 (-/-) progeny [23
].
Most studies to understand how TGF-ß family members elicit their biological effects have been performed on adherent cells, such as epithelial, endothelial, and mesenchymal cell lines [1
, 2
]. Through these investigations, the intracellular pathways of TGF-ßs, activins, and BMPs have been characterized in considerable detail. TGF-ß family members bind to specific heteromeric transmembrane receptor complexes with intrinsic serine/threonine kinase activity. These receptor complexes transduce the signal intracellularly via effector proteins termed Smads. Upon activation, the Smad proteins accumulate in the nucleus, where they participate in the regulation of transcription of target genes [24
25
26
]. Here, we review how the potent biological effects of TGF-ß on immune cells can be explained by our current knowledge of the TGF-ß signal transduction pathway.
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TGF-ß EFFECTS ON IMMUNE CELLS
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TGF-ß modulates the proliferation, differentiation, and function of all classes of lymphocytes, macrophages, and dendritic cells, thus regulating the innate, nonantigen-specific as well as the antigen-specific immunity [6
7
8
]. It is important that effects of TGF-ß are dependent on the cell type and its differentiation state as well as on the combination of cytokines that is present in the medium [6
7
8
]. Thus, it is difficult to understand the implication of TGF-ß in immune cells using in vitro experiments. However, as has become evident from TGF-ß1 knockout mice, TGF-ß plays a pivotal role in the maintenance of immune cell homeostasis. TGF-ß1 null mice that are born die shortly after weaning as a result of multifocal, inflammatory disease with lymphocyte infiltration into multiple organs [21
, 22
] and autoimmune manifestations [27
, 28
]. Detailed analysis of these mice revealed that there is an increased adhesion of leukocytes to the vascular endothelium and increased levels of major histocompatibility complex (MHC) molecules at the onset of inflammation [29
, 30
]. The phenotype of these mice can be attributed, in part, to a loss of the antiproliferative effect of TGF-ß1 on lymphocytes. These studies clearly implicate endogenous TGF-ß1 in the suppression of autoimmunity. Mice deficient in TGF-ß2 or TGF-ß3 have severe developmental defects and die before they form any immune cell and are thus not informative as to whether these isoforms are important in the regulation of immune cells [12
].
In vitro studies have shown that TGF-ß affects B and T cells at all stages of development, and biological response is strongly dependent on cellular context. Typically, however, TGF-ß inhibits B- and T-cell proliferation and stimulates apoptosis, thus acting as an immunosuppressive molecule in accordance with the phenotype of the TGF-ß1 knockout mouse [6
7
8
]. Recently, TGF-ß1 was proposed to suppress inflammation by promoting the death of postactivated T cells [31
]. Furthermore, apoptotic T cells release TGF-ß, thereby contributing to an immunosuppressive environment [32
]. It is interesting that TGF-ß1 can inhibit the growth and survival of many transformed lymphocyte progenitors, including a subset of chronic lymphocytic leukemia and acute lymphoblastic leukemia cells. BMP-2, -4, and -7 and activins have the same effects on hybridomas and multiple myeloma cell lines [33
34
35
].
TGF-ß has also been shown to play a role in T-helper (Th) subset differentiation. Again, the cytokine environment influences the effects of TGF-ß to a great extent. However, there is a tendency of the TGF-ß effect to promote the differentiation of CD4(+) cells into Th1-like cells. Thereby, TGF-ß shifts the profile toward Th1 compared with Th2 cytokines and induces the production of interleukin (IL)-2 and, depending on the activation factor and the source of T cells, interferon-
(IFN-) [7
]. However, differential responses in naïve and memory T cells have been observed; Ludviksson et al. [36
] demonstrated that TGF-ß inhibits Th1/Th2 responses in naïve T cells but inhibits only Th1 response in memory T cells. It remains controversial whether TGF-ß acts directly or indirectly by counteracting other differentiation pathways [7
].
TGF-ß is shown to play a role in antibody class-switching. Antibody class-switch recombination occurs after antigen activation of B cells. During this process, the region of an immunoglobulin (Ig) heavy-chain gene, which encodes the antigen-recognizing portion, is recombined with the constant region of a different Ig class. TGF-ß1 directs class-switch recombination in Ig isotype IgA as a result of its ability to induce transcription from germline Ig genes [6
, 8
]. Of note, mice lacking TGF-ß1 are partially IgA-deficient [37
]. TGF-ß also controls several aspects of the normal maturation and differentiated functions of B cells [6
, 8
]. This includes the regulation of the expression of cell-surface molecules, such as inhibition of IgM, IgD, CD23, and the transferrin receptor, and the induction of MHC class II expression on pre-B cells and mature B cells [6
, 8
].
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SIGNAL TRANSDUCTION ACROSS THE PLASMA MEMBRANE
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Structure and activation of TGF-ß receptors (TßRs)
TGF-ß family members initiate intracellular signaling by inducing the assembly of a heterotetrameric complex of two types of transmembrane receptors known as type I and type II receptors [1
, 2
]. Types I and II have an N-glycosylated extracellular domain that is rich in cysteine residues, one transmembrane domain, and an intracellular serine/threonine kinase domain (Fig. 2
). The type II receptor kinase is a constitutively active kinase, whereas the type I receptor kinase needs to be activated by the type II receptor kinase. Upon ligand-induced formation of the heteromeric complex, the type II receptor phosphorylates the type I receptor in a region rich in glycine and serine/threonine residues (termed the GS domain) [38
]. This phosphorylation changes the conformation of the type I receptor, thereby activating its kinase. The activated type I receptor then propagates the signal by phosphorylating specific intracellular proteins (Fig. 2) . Thus, the type I receptor acts downstream of the type II receptor and consistent with this notion, has been shown to determine signaling specificity [1
, 2
]. Several type I (also called activin receptor-like kinase, ALK) and type II receptors have been identified. TGF-ß, activin, and BMP signal each via a distinct set of type I and type II receptors [1
, 2
]. On most cell types, TGF-ß binds first to TßR type II (TßR-II) and subsequently recruits TßR-I (ALK-5) [38
39
40
]. ALK-1 is a TßR-I that is expressed specifically in endothelial cells [41
]. Initially, activin binds to an activin type II receptor (ActR-II or ActR-IIB) and subsequently recruits ActR-I (ALK-2) or, more frequently, ActR-IB (ALK-4) [42
43
44
]. BMP binds to a type II receptor (ActR-II, ActR-IIB, or BMPR-II) and a type I receptor (ALK-2, ALK-3, or ALK-6) in a cooperative manner [45
, 46
].
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Figure 2. Activation of TßRs. Ligand binds initially to TßR-II. This can be recognized by TßR-I, which is then recruited into the complex. Subsequently, the constitutively active TßR-II kinase phosphorylates and activates the TßR-I, which propagates the signal downstream.
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In addition to the signaling type I and type II receptors, TGF-ß can bind to receptor-associated transmembrane proteins, such as betaglycan (also called TßR-III) and endoglin [47
, 48
]. These receptors do not have any intrinsic enzymatic activity but have been shown to modulate TGF-ß signaling. TßR-III can facilitate the binding to TßR-II [47
]. The mechanism by which endoglin affects TßR signaling is still unclear.
Expression and in vivo function of TßRs
In many studies, biological response of immune cells to TGF-ß1 was studied without knowing the expression of its receptors. However, hematopoietic cells, including macrophages and B-cell precursors, have been shown to express TßR-I and TßR-II [49
]. In vitro activation of resting B cells results in an increase of TGF-ß ligand and receptor expression, which may result in a self-limitation of B-lymphocyte clonal expansion and ultimate differentiation [50
]. In addition, leukemic B-cell precursors, which are growth-inhibited by TGF-ß1, were also found to express TßR-I and TßR-II [51
]. By performing conditional mutagenesis of TßR-II in mice, differential roles of TßR-II in homeostasis and antigen responsiveness of B-cell subpopulations have been revealed [52
]. Mice selectively lacking TßR-II in B cells show a reduced lifespan of the conventional B cells, expansion of peritoneal B-1 cells, B-cell hyperplasia in Peyer’s patches, elevated serum Ig, and substantial IgG3 responses to a normally weak immunogen. This B-cell hyperresponsiveness is associated with a virtually complete serum IgA deficiency and the generation of autoreactive DNA-binding antibodies [52
]. Expression of a dominant-negative TßR-II under a T-cell-specific promoter demonstrates that in the absence of TGF-ß signaling, most of the T cells differentiate spontaneously into Th1/Th2 cytokine-secreting cells, indicating that T-cell homeostasis also requires TGF-ß signaling. These mice, like TGF-ß1 knockout mice, develop autoimmune manifestations [53
]. Taken together, these studies point out the importance of an active signaling cascade involving the TßR-II in T- and B-cell homeostasis.
Betaglycan and endoglin mRNA are present in leukemic B-cell precursors of patients with common acute lymphoblastic leukemia. However, the involvement of these receptors in the disease has not been investigated [51
].
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INTRACELLULAR SIGNALING
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Structure and activation of Smad proteins
Type I receptors initiate intracellular signaling by phosphorylating specific proteins known as Smad proteins (for Sma and Mad proteins from Caenorhabditis elegans and Drosophila, respectively) [24
25
26
]. Smad proteins can be divided into three distinct classes: the receptor-activated Smads (R-Smads), the common-mediator Smads (Co-Smads), and the inhibitory Smads (I-Smads; Fig. 3a
). Smad proteins share two highly conserved domains, Mad-Homology domains 1 and 2 (MH1 and MH2) at N- and C-terminal parts of the proteins, respectively. A divergent proline-rich region of variable length adjoins these two domains. Cytosolic R-Smads transiently interact via their MH2 domains with a specific, activated type I receptor and become phosphorylated at their extreme C-terminal serine residues [54
, 55
]. Whereas Smad1, Smad5, and Smad8 are mainly involved in BMP signaling, Smad2 and Smad3 are restricted to the TGF-ß/activin pathway [56
57
58
]. Upon activation, R-Smads form heteromeric complexes with the Co-Smads (i.e., Smad4 in mammals) via their MH2 domains. The number of Smads and the stoichiometry between R-Smads and Co-Smads in the heteromeric complex are unclear; complexes with one R-Smad and one Co-Smad [59
] and two R-Smads and one Co-Smad have been proposed [60
, 61
]. The R-Smad/Co-Smad heteromeric complex accumulates in the nucleus, where it participates in the control of expression of target genes (see below). Inhibitory Smads (i.e., Smad6 and Smad7) prevent the activation of signal-transducing R- and Co-Smads, and several mechanisms for their inhibitory action have been proposed. I-Smads interact efficiently with activated type I receptors and compete with R-Smads for binding to the activated type I receptor [62
63
64
]. In addition, Smad6 has been shown to compete with Smad4 for interacting with activated Smad1 [65
]. Furthermore, Smad7 has been found to interact constitutively with HECT-domain ubiquitin ligases, termed Smurf1 and Smurf2 [66
, 67
]. Upon recruitment of the Smad7/Smurf complex to the activated TßR, Smurf induces TßR degradation through proteosomal and lysosomal pathways.
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Figure 3. The TGF-ß/Smad pathway. (a) The Smad family can be divided in three distinct subgroups: R-Smads, Co-Smads, and I-Smads. Schematic structures of R-Smads, Co-Smads, and I-Smads are indicated. MH1 and MH2 domains are N- and C-terminal regions of high similarity, respectively. The ß-hairpin loop, which is important for interaction with DNA, is indicated. The serine residues in the C-terminal SXS motif in R-Smads can be phosphorylated by activated type I receptors. (b) Smad activation. Upon ligand-induced heteromeric complex formation and activation of type I receptor kinase, R-Smads are phosphorylated. These activated R-Smads form heteromeric complexes with Co-Smads and accumulate in the nucleus. Together with coactivators (p300, CBP, P/CAF), corepressors (TGIF, SnoN, c-Ski, or CtBP), and transcription factors (TF; e.g., FAST, mixer, milk, AML), these Smad complexes participate in transcriptional regulation of target genes.
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Regulation of transcription by Smads
Smad proteins do not contain any intrinsic enzymatic activity but rather exert their function through protein-DNA and protein-protein interactions via their MH1 and MH2 domains (see below). MH1 and MH2 domains of R-Smads have intrinsic affinity for each other and inhibit each other’s functions [68
]. The ligand-triggered C-terminal serine phosphorylation in R-Smads relieves this autoinhibition [1
, 2
] and allows R-Smads to interact with DNA and other proteins. Smad3 and Smad4 MH1 domains interact directly with a 5' GTCT DNA sequence known as the Smad-binding element (SBE; Fig. 3b
) [69
70
71
]. Structural determination of the Smad3 MH1 domain with SBE revealed that a protruding ß-hairpin loop, which is highly conserved in all R- and Co-Smads, is responsible for direct DNA contact [71
]. SBEs have been shown to be important for TGF-ß-induced activation of several genes [24
25
26
]. Smad2, in contrast to its highly similar Smad3, does not bind directly to DNA. Smad2 has an extra loop compared with Smad3, which is in close proximity to the putative DNA-binding motif in Smad2; this extra sequence in Smad2 interferes with DNA binding [71
72
73
]. Smad3, Smad4, and BMP-activated R-Smads have also been shown to bind directly to GC-rich sequences. However, more studies are needed to determine whether this is of true physiological significance [74
].
The binding of Smads to DNA occurs with rather low affinity [71
] and sequence specificity [74
]. Therefore, Smads need to cooperate with each other and/or with other DNA-binding proteins to regulate TGF-ß target gene transcription (Fig. 3b) . Such cooperation has been shown to occur with several transcription factors and can take place via their MH1 domain or MH2 domains [24
25
26
]. A Smad-interacting motif, containing the PPNK sequence, has been found to mediate the interaction of Smads with winged helix/forkhead proteins FAST1/2 and paired-like homeodomain proteins mixer/milk [76
, 77
].
Smad proteins have transcriptional activity when fused to a heterologous DNA-binding domain and stimulated with ligand [78
]. The mechanistic explanation for this activity was provided by the capacity of the MH2 domain to recruit transcriptional coactivators, such as CBP/p300 or P/CAF, in a ligand-dependent manner (Fig. 3b) [79
80
81
82
83
]. These proteins possess an intrinsic histone acetyltransferase (HAT) activity. Acetylation of lysine residues in the N-terminal tails of histones facilitates gene activation, presumably by reducing histone affinity for the DNA, thereby promoting the binding of transcription factors to nucleosomal DNA. Overexpression of these coactivators favors Smad-induced transcription. Alternatively, Smad proteins interact with transcriptional corepressors, which recruit histone deacetylases (HDAC) to Smad complexes, thereby repressing Smad transcriptional activities. Ski, SnoN, and TGIF are such corepressors that interact with the MH2 domain of Smad proteins (Fig. 3b)
[84
85
86
]. TGIF can interact directly with HDAC, whereas c-Ski and SnoN interact indirectly via proteins such as N-CoR and Sin3A. The balance between coactivators and corepressors at the site of transcription might determine whether Smad might activate or repress gene transcription. It is interesting that SnoN has been proposed to keep TGF-ß target genes off when cells are not stimulated with TGF-ß [86
]. The rapid TGF-ß-induced degradation of SnoN via Smad-mediated recruitment of Smurf2 may ensure that TGF-ß/Smad-induced gene responses can proceed [87
]. Of note, a number of negative regulators of TGF-ß/Smad signaling, such as SnoN and I-Smads, are induced by TGF-ß or other stimuli [88
89
90
91
]. Thus, these negative regulators may participate in negative feedback control and crosstalk with other signaling pathways and regulate the intensity and duration of TGF-ß signaling responses.
TGF-ß/Smad signaling in immune cells
In vivo data support the importance of Smad proteins in immune cells. Smad3-deficient mice exhibit chronic infection, revealing that Smad3 plays an important role in the TGF-ß-mediated regulation of T-cell activation and mucosal immunity [92
]. In addition, neutrophils and monocytes are largely absent in the early wound of these Smad3 null mice, suggesting that Smad3 is also critical for TGF-ß-mediated chemotaxis of inflammatory cells [93
]. Indirect evidence of the involvement of Smads in TGF-ß maintenance of T-cell homeostasis also comes from transgenic mice that selectively express Smad7 in mature T cells [94
]. Although these mice do not show a specific phenotype spontaneously, antigen-induced airway inflammation and airway reactivity are enhanced. These observations correlate with the high production of Th1 and Th2 cytokines. Therefore, regulation of T cells by TGF-ß appears crucial for the negative regulation of the inflammatory (immune) response [94
].
An example of TGF-ß1-induced transcriptional regulation via Smads is IgA antibody class-switch recombination in splenic B cells. TGF-ß is critically important for the IgA class-switch to occur and cannot be substituted by other factors in vivo. Acute myelogenous leukemia 1 (AML1) and AML2 transcription factors, consisting of different -subunits and a common ß-subunit, control this class-switching by activating the germline IgA1 and IgA2 promoters via an I enhancer element [95
]. The presence of several SBEs was noted in this regulatory element. Smad3 and Smad4 were found to bind to these SBEs and to cooperate with the AML transcription factors to activate this I promoter element in a TGF-ß-dependent manner [96
, 97
]. The -subunits of AML were found to interact with Smads mainly via the MH2 domains. Mutations of AML and/or SBE binding sites inhibit TGF-ß-induced activation of this promoter [96
, 97
]. Recently, Ets proteins and cAMP response element binding protein (CREB) have also been implicated in TGF-ß-mediated I promoter activation [95
, 98
, 99
]. Thus, an entire set of transcription factors may cooperate to orchestrate the TGF-ß-induced IgA class-switching.
An example of a gene that is transcriptionally repressed by TGF-ß is GATA-3 [100
, 101
]. This Th2-specific transcription factor is implicated in the expression of the IL-5 gene by interacting directly with a critical regulatory element of its promoter. TGF-ß has been shown to reduce CD4(+) cell differentiation into Th2 cells by inhibiting GATA-3 expression. Ectopic expression of this zinc finger transcription factor in developing T cells overcomes the ability of TGF-ß to inhibit Th2 differentiation [100
, 101
]. Whether Smads are involved in repression of this gene remains to be investigated.
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TGF-ß-INDUCED GROWTH ARREST
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In many cell types, TGF-ß causes a G1 cell-cycle arrest by inhibiting cyclin-dependent kinase (CDK) activity via activation of p15INK4b and p21Cip1 expression [1
, 2
]. In a mouse B-cell hybridoma cell line that is potently inhibited by BMP-2, BMP-2 was observed to increase the level of the p21Cip1 protein and to maintain pRb protein in its hypophosporylated state (Fig. 4
) [102
]. Recently, the p21Cip1 promoter was found to contain an SBE, capable of binding Smad1 and Smad4, which was shown to be critically important for the BMP-induced activation of this promoter [103
].
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Figure 4. TGF-ß can induce a G1 cell-cycle arrest. Cyclin-dependent kinase activity is required for cell-cycle progression toward the S phase. As TGF-ß promotes the expression of cyclin-dependent kinase inhibitors (CDKI), such as p15INK4b or p21Cip1, it inhibits the kinase activity associated with cyclin-CDK complexes, thereby maintaining the retinoblastoma protein (Rb) in its hypophosphorylated state. This form of Rb interacts and inhibits the E2F transcription factor, transcription of gene implicated in the S phase entry is thus blocked, and cells are arrested in the G1 phase. TGF-ß also inhibits the expression of c-myc protein. c-myc can repress the expression of CDKI. Thus, TGF-ß-induced repression of c-myc expression favors the production of CDKI.
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In a recent study, normal and transformed lymphocyte progenitors were shown to be growth-arrested by TGF-ß1 via up-regulation of the Id3 protein, an inhibitor of E-basic helix-loop-helix transcription factor activity [104
]. In cells lacking Id3, the TGF-ß-induced growth arrest and apoptosis are perturbed, establishing Id3 as an important downstream TGF-ß target in these cells. This is likely a Smad-dependent effect, because ectopic expression of Smad7 inhibits the TGF-ß-induced Id3 expression.
TGF-ß also reduces the production of survival cytokines, thereby inhibiting cell proliferation indirectly. For example, TGF-ß1 inhibits IL-2- and IL-12-induced cell proliferation and IFN- production by T and natural killer (NK) cells, for which there are conflicting studies on the mechanism involved. In one study, TGF-ß1 was shown to inhibit IL-2-induced tyrosine phosphorylation and activation of JAK1 and STAT5 in murine T lymphocytes [105
]. In addition, in human T cells activated by allogeneic MHC molecules, TGF-ß1 was found to inhibit IL-12-induced phosphorylation of JAK2, Tyk2, and STAT4 [106
]. In primary T cells and an NK cell line, however, TGF-ß1 was shown to have no effect on IL-2- or IL-12-induced activation of JAK and STAT proteins [107
], suggesting an alternative mechanism of inhibition by TGF-ß.
TGF-ß might have a role to play in the maintenance of the unstimulated T-lymphocyte quiescent state, as suggested by a recent study [108
]. Tob, a member of the Tob and BTG antiproliferative protein family, prevents T-cell cycle progression and blocks IL-2 transcription through its interaction with Smad2 and Smad4. This interaction promotes the binding of Smad proteins to a negative regulatory element in the IL-2 promoter. Thus, TGF-ß might block unstimulated T-cell proliferation indirectly by inhibiting IL-2 production [108
].
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TGF-ß-INDUCED APOPTOSIS
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TGF-ß family members can induce apoptosis in several cell types, including B cells and fully differentiated plasma cells [102
, 109
]. Often, TGF-ß-induced apoptosis is accompanied with growth inhibition. TGF-ß-induced apoptosis can be mediated via Smad proteins, because ectopic expression of Smads has been shown to enhance TGF-ß-induced apoptosis in certain cells [110
, 111
]. Regulation of the expression of pro- and antiapoptotic Bcl family members has been implicated in TGF-ß-induced apoptosis (Fig. 5
) [112
, 113
]. In an immature B-cell line, TGF-ß1-induced apoptosis is preceded by a decline in c-myc expression. This repression has been associated with the inhibition of the nuclear factor-
B (NF-B)/Rel activity via up-regulation of IB, which sequesters NF-B/Rel in the cytoplasm [109
]. However, whether Smads are (directly) mediating the regulation of expression of these genes involved in apoptosis is not known.
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Figure 5. TGF-ß can trigger programmed cell death. Several mechanisms for TGF-ß-induced apoptosis have been proposed, including the JNK/p38 cascade and/or the modulation of the balance between anti- and proapoptotic proteins of the Bcl family.
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TßR-initiated pathways, distinct from the Smad pathway, have also been implicated in TGF-ß-induced apoptosis. TGF-ß induces the activation of the TGF-ß-activated kinase 1 (TAK1), a protein of the mitogen-activated protein kinase (MAPK) kinase kinase family that can activate the p38 pathway (Fig. 5)
[114
]. This TAK1-p38 pathway has been shown to be involved in the BMP-2-induced apoptosis in an IL-6-dependent mouse hybridoma [35
]. Activation of the BMP pathway leading to apoptosis can be inhibited by overexpression of the inhibitory Smad6 [35
]. Recently, Daxx, a Fas-receptor-associated protein, has been proposed to act as a mediator of TGF-ß1-induced apoptosis in a murine immature B-cell lymphoma cell line via its interaction with TßR-II. Daxx acts downstream of TßR-I and activates the JNK pathway [115
].
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TGF-ß AND CANCER
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TGF-ß has a biphasic role in tumorigenesis. In early phases, when cells still respond to the growth inhibitory effects of TGF-ß, it may act as tumor suppressor. However, in late phases, when cells have escaped selectively from the antimitogenic response of TGF-ß, and tumor cells often start to secrete high amounts of TGF-ß, it may act as a tumor promoter [116
, 117
].
In accordance with the proposed role in tumor suppression for TGF-ß intracellular components, the genes encoding TßRs and Smads have been found genetically altered in human cancers. In particular, TßR-II is frequently mutated in colon and gastric cancers with a microsatellite instability phenotype [118
]. In addition, mutated TßRs or down-regulation of receptor expression have been observed in lymphocytic leukemia and cutaneous T-cell lymphoma [119
120
121
122
]. The frequent mutation and homozygous deletion of smad4 in pancreatic cancers led to its original discovery as the "deleted in pancreatic cancer" (DPC4) tumor suppressor gene [123
]. Subsequently, Smad4 was also found mutated in other types of cancers, albeit less frequently than in pancreatic cancers. In acute myelogenous leukemia, missense mutations in Smad4 were identified that cause a single amino acid change in the MH1 domain and a frame-shift mutation resulting in a truncated version of the protein [124
]. These mutants have lost the ability to activate transcription. Furthermore, they act as dominant-negative inhibitors of the wild-type protein by blocking the DNA binding or nuclear translocation of wild-type Smad4.
Whereas mutations in smad2 have also been observed in colorectal and lung cancers [125
, 126
], no mutation in smad3 has been observed in human malignancy. However, in certain tumors, there may be dysregulation of Smad3 function. In chronic myeloid leukemia, TGF-ß signaling is blocked by an abnormal expression of Evi-1, a zinc-finger oncoprotein that interacts with Smad3. Evi-1 has been shown to abrogate the binding of Smad3 to DNA [127
] and to repress Smad-induced transcription by recruiting the corepressor C-terminal binding protein (CtBP) [128
]. Chromosomal translocations involving the c-Ski protooncogene have been frequently associated with non-Hodgkin’s lymphoma and pre-B acute lymphoblastic leukemia [129
, 130
]. Constitutively expressed c-Ski may repress Smad3 activity in these hematopoietic malignancies. In accordance with the TGF-ß-mediated evasion of tumor from immune response, a recent study shows that the abrogation of TGF-ß signaling leads to the enhancement of anti-tumor immunity [131
]. Transgenic mice expressing a dominant-negative TßR-II in T cells only develop an immune response capable of eradicating tumors when the mice are challenged with live tumor cells [131
].
In late-stage tumors, high amounts of TGF-ß produced by tumors can stimulate tumorigenesis. TGF-ß can do this directly by promoting tumor-cell invasion and metastasis [117
]. In this case, the tumor cells have selectively escaped the growth inhibitory response and remain sensitive to TGF-ß in certain other aspects. TGF-ß can also stimulate tumorigenesis indirectly by promoting angiogenesis and tumor stroma formation and by its potent immunosuppresive action [116
, 117
]. In this respect, it is interesting to note that glioblastoma patients that are severely immunosuppressed show marked elevated expression of TGF-ß2. Antisense oligonucleotide-mediated inhibition of TGF-ß2 was found to reverse the cellular immunosuppression in malignant glioma [132
] and may make glioblastoma patients more responsive to immunotherapy [133
].
|
PERSPECTIVES
|
|---|
In vitro effects of TGF-ß on leukocytes are highly context-dependent. In vivo investigations favor the immunosuppressive activity of TGF-ß, as demonstrated by the multifocal inflammatory phenotype of TGF-ß1-deficient mice. The interpretation of the TGF-ß1 knockout is complicated by the fact that the effects are very severe and involve multiple cell types. Certain less severe effects may be obscured, and effects that occur late in life are missed because of death after weaning. Thus, for further understanding of the in vivo importance of TGF-ß, creation of mice, which are deficient in TßR or Smad signaling in only one particular immune cell type and/or in a particular time window, has and will continue to be extremely informative [52
, 53
, 92
, 131
]. Compared with ligand knockouts, these mice have the advantage that the effects are cell-autonomous and, for example, allow one to dissect the role of the different cell types in the inflammatory response in TGF-ß1-deficient mice.
Elucidating the role of TGF-ß in immune cells will be important to improve our understanding of the several immune diseases that are caused by subversion in TGF-ß signal transduction or that show a perturbation in TGF-ß signaling of which the pathological significance has not yet been established. Whereas most studies on immune cells have been performed with TGF-ß1, other TGF-ß family members are also expected to have potent effects. This is an exciting area for future research.
|
ACKNOWLEDGEMENTS
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|---|
S. D. is supported by TMR EC network grant (ERB FMRX-CT98-0216). M-J. G. is supported by the Netherlands Organization for Scientific Research (MW902-16-295).
We are grateful to Alexander Rosendahl and Jurgen Roes for valuable comments.
Received September 4, 2001;
revised February 1, 2002;
accepted February 3, 2002.
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ABSTRACT
INTRODUCTION
