ANTICANCER RESEARCH 28 : 1531-1540 (2008)

Review

TGFβ-regulated Gene Expression by Smads and Sp1/KLF-like Transcription Factors in Cancer VOLKER ELLENRIEDER

Signal Transduction Laboratory, Internal Medicine, Department of Gastroenterology and Endocrinology, University of Marburg, Marburg, Germany

Abstract. Transforming growth factor beta (TGF β) controls complex induces the canonical Smad signaling molecules which vital cellular functions through its ability to regulate gene then translocate into the nucleus to regulate transcription (2). The expression. TGFβ binding to its transmembrane receptor cellular response to TGF β can be extremely variable depending kinases initiates distinct intracellular signalling cascades on the cell type and the activation status of a cell at a given time. including the Smad signalling and transcription factors and also For instance, TGF β induces growth arrest and apoptosis in Smad-independent pathways. In normal epithelial cells, TGF β healthy epithelial cells, whereas it can also promote tumor stimulation induces a cytostatic program which includes the progression through stimulation of cell proliferation and the transcriptional repression of the c-Myc oncogene and the later induction of an epithelial-to-mesenchymal transition of tumor induction of the cell cycle inhibitors p15 INK4b and p21 Cip1 . cells (1, 3). In the last decade it has become clear that both the During carcinogenesis, however, many tumor cells lose their tumor suppressing and the tumor promoting functions of TGF β ability to respond to TGF β with growth inhibition, and instead, are primarily regulated on the level of gene expression through activate genes involved in cell proliferation, invasion and Smad-dependent and -independent mechanisms (1, 2, 4). In metastasis. Strong efforts have been made during recent years to many cases, receptor activation not only stimulates the characterize Smad-mediated transcriptional processes and to downstream Smad cascade, but also Smad independently identify those TGFβ-regulated transcription factors that control signalling and transcription molecules which might work gene expression independent of the Smads. These studies have together or independent from each other to regulate gene led to the identification of a novel family of TGFβ-inducible expression in response to TGF β (3, 4). In addition, TGF β Sp1/KLF-(Krüppel-like factors) like transcription factors induces the expression of early response transcription factors (KLF10 and KLF11) which play remarkable roles in TGFβ such as the Sp1/KLF-(Krüppel-like-factors) like zinc-finger mediated cell growth control and differentiation. In this article, proteins KLF10 and KLF11 which work as effector proteins in the current knowledge on the peculiar roles of Sp1/KLF-like TGF β mediated cell growth control and differentiation (5, 6). proteins in Smad dependent and -independent gene regulation Following induction by TGF β, KLF11 represses the expression initiated by TGFβ, are summarized. of the c-Myc oncogene, terminates the negative Smad7 feedback loop and renders cells more sensitive to oxidative drugs through Transforming growth factor- β (TGF β) is a pleiotropic cytokine silencing of the oxidative scavengers superoxide dismutase 2 that regulates a wide variety of cellular processes including cell (SOD2) and Catalase1 (7, 8). Taken together, the recent studies growth and apoptosis, differentiation, migration, and metastasis suggest a model in which the rapid induction of KLF11 (1). TGF β binding to its transmembrane receptor kinase contributes to TGF β-induced cell growth inhibition through different transcriptional mechanisms including concerted actions with the Smads but also Smad-independent mechanisms.

Correspondence to: Volker Ellenrieder, MD, Department of Internal TGF β – Activation of Effector Signalling and Medicine, Gastroenterology and Endocrinology, University of Transcription Pathways Marburg Baldinger Strasse 35043 Marburg, Germany. Tel: +49 0 64212866460, Fax: +49 0 64212868922, e-mail: [email protected] marburg.de The prototype of the TGF β family, TGF β1, binds with high affinity to the type-II TGFβ receptor (TβR-II), which then Key Words: TGF β, Smads, Sp1/KLF-like transcription factors, gene recruits the type-I TGFβ receptor (TβR-I) to form a expression, review. heteromeric complex (1, 9). This three-component module

0250-7005/2008 $2.00+.40 1531 ANTICANCER RESEARCH 28 : 1531-1540 (2008) then allows the kinase activity of the type-II receptor to stimulation, Smads constantly undergo cycles of receptor- phosphorylate the TGFβ receptor and subsequently to bind mediated phosphorylation (activation) and phosphatase- signalling molecules such as members of the Smad family of mediated dephosphorylation, resulting in sustained nuclear- transcription factors (9). Although there are considerably cytoplasmic shuttling of the transcription factors (15). fewer receptors and Smads than there are ligands, a greater Receptor-mediated Smad activation and nuclear translocation versatility of signalling is possible than might be expected. is finally terminated by inhibitory Smads, Smad6 and Combinatorial interactions of type I and type II receptors and Smad7, which serve as negative feedback loops in TGFβ Smads in oligomeric complexes allow substantial diversity, signalling (16). Upon induction by TGFβ or other growth and are complemented by the many sequence-specific factor signals, Smad7 binds to the activated type-I TGFβ transcription factors with which Smads cooperate, resulting receptor in competition with R-Smads and thereby blocks in context-dependent transcriptional regulation (2, 10). In further activation and downstream signal transduction of the addition to the Smad signaling pathway, activation of the Smads (16). receptor complex initiates signalling through Smad Genetic alterations of this simple TGF β signalling independent mechanisms (4). These alternative, non-Smad pathway are frequently found in somatic and heritable pathways can either work in concert with or independent disorders as well as in various tumor entities. Inactivating from Smad proteins to regulate vital cellular functions mutations of the type-II receptor, for instance, have been through transcriptional responses to TGFβ (4, 7, 10). Finally, reported in colorectal, gastric and endometrial carcinomas TGFβ can induce immediate early response transcription and in association with a more aggressive phenotype (17). factors, such as the KLF10 and KLF11 zinc-finger proteins, In addition, more than half of pancreatic carcinomas and which function as nuclear regulators to repress or activate approximately one third of colorectal carcinomas harbor gene expression together with or irrespective of the Smad inactivating mutations of the Smad4 gene based on proteins (5, 6). homozygous deletion or intragenic mutation (18-20). In many cases, genetic loss of Smad4 causes disrupted The Smad Signalling Pathway in Normal nuclear-cytoplasmic shuttling of Smad complexes and thus and Malignant Epithelial Cells renders tumor cells insensitive to nearly all TGFβ-regulated transcriptional responses, including those associated with Smad proteins are central downstream effectors of TGF β growth inhibition. Genetic alterations of the Smad4 gene signaling in normal and transformed cells. Smads exist as are also found in familial juvenile polyposis, an autosomal three subgroups: Receptor-Smads ( e.g. Smad2 and Smad3), dominant disease characterized by predisposition to common Smads ( e.g. , Smad4), and inhibitory Smads (Smad6 gastrointestinal polyps and cancer (21). In this syndrome, and Smad7) (9). Smad2 and Smad3 serve as receptor- polyps are formed by inactivation of the Smad4 gene activated signalling molecules which transduce the TGFβ- through germline mutation and loss of the unaffected wild- stimulus into the nucleus following interaction with Smad4. type allele. Together, disruption of TGFβ-Smad signalling Inactive R-Smads are anchored to the plasma membrane through inactivating mutations is commonly observed in through various molecules, among which the Smad anchor different malignancies and is closely associated with the for receptor activation (SARA) has been most extensively loss of tumor suppression by TGFβ (22). Importantly, studied (11). Upon ligand-binding, the activated TGFβ type- however, signal transduction and target gene transcription I (T βR-I) kinase phosphorylates the C-terminal Ser-Ser-X- by the Smads is not only affected by genetic alterations Ser motif of R-Smads, allowing them to form heteromeric within the pathway, but also through signalling crosstalk complexes with Smad4 and to move into the nucleus in order interactions. In fact, at one level or another, essentially all to regulate the expression of selected TGFβ target genes (9, major tumor suppressor and oncogenic signaling pathways 11, 12). X-ray crystallographic studies have shed much light can converge on Smads as a node for signal integration and on the mechanisms of how Smad proteins interact with each thus can positively or negatively influence Smad-mediated other (12). Structurally, Smad proteins consist of two “Mad- transcription (2, 10, 14). Inputs by other pathways occur at homology” domains, MH1 and MH2, which are connected the level of receptor activation, R-Smad complex formation by a flexible linker region. The MH1 domain causes DNA- with Co-Smad4, or at the level of the nuclear translocation binding, whereas the MH2 domain is responsible for of pre-formed R-Smad/Co-Smad complexes (2, 10, 13, 23). physical interactions with Smad partnering transcription Crosstalks between Smads and distinct mitogen-activated factors to assemble transcriptional complexes (10, 13, 14). protein kinases (MAP kinases) such as extracellular signal- Nuclear Smad proteins are targets of certain protein regulated kinases (Erk) 1 and 2 and the stress-activated phosphatases and ubiquitin ligases resulting in their protein kinases, Jun N-terminal kinase (JNK) and p38, are reversible return to the cytoplasm for another round of of particular interest in carcinogenesis (13, 23, 24). For receptor-mediated phosphorylation. Thus, under TGF β instance, mutational activation of MAP kinase pathways is

1532 Ellenrieder : Sp1/KLF-like Proteins and Smad Gene Regulation Initiated by TGF β ( Review) frequently found in human carcinomas, most notably via unable to mount the TGFβ gene response that depends on the activation of oncogenic Ras, an upstream activator of that particular R-Smad complex combination (14). the proliferative Raf-Mek-Erk signaling cascade, which is Moreover, in some cases Smad partners also determine the an early event in many tumors, such as pancreatic cancer direction of the effect, activation or repression, that is exerted (25, 26). Persistent activation of MAP kinase pathways by on a target gene. Smad partners therefore provide four levels oncogenic Ras inhibits Smad signaling through Erk- of specificity: target gene specificity, pathway specificity, cell mediated phosphorylation of MAP kinase sites within the type specificity and the specific transcriptional effect. The linker region of Smad2 and Smad3, thereby blocking the number of DNA-binding transcription factors with which complex formation with Smad4 (13). The linker region is a Smads can functionally interact is large, and these proteins critical phosphorylation site for several kinase regulated are also often regulated by multiple signalling pathways, signaling pathways. For instance, Smad proteins contain including those that were mentioned above. This is 2+ multiple phosphorylation sites for Ca /calmodulin- particularly important in cancer cells, in which the dependent protein kinase II (CamKII) and protein kinase C transcriptional response to TGFβ is profoundly impaired due (PKC), which phosphorylate Smad2 and Smad3 and to cytoplasmic crosstalks and altered expression/activation of thereby inhibit either the nuclear translocation or the DNA- Smad-DNA partner transcription factors. binding of Smads (14). In contrast, signaling crosstalk with Through the recruitment of transcriptional coactivators or JNK or MEKK1, an upstream activator of the JNK corepressors to target promoters, Smad transcriptional pathway, has been shown to facilitate the nuclear complexes can either induce or inhibit target gene expression. accumulation of Smad complexes and thereby accelerate The first identified Smad-interacting transcription factor was Smad-signaling in cancer cells (24). Together, the Smad the forkhead family member forkhead box class H1 (FoxH1) signaling transduction pathway is commonly altered by (28). Three other forkhead family members, forkhead box class either genetic alterations or crosstalk interactions with O, FoxO1, FoxO3, and FoxO4, serve as Smad3 transcriptional cascades that play important functions in gene regulation partners in the activation of the cyclin-dependent kinase during carcinogenesis (Figure 1). inhibitor p21Cip1 or CDKN1A (29). The Runt-related (Runx) gene family of DNA-binding factors comprises three members Smad-dependent Transcription in that interact with the Smads to mediate TGFβ-regulated Normal and Malignant Epithelial Cells transcription: Runx1, Runx2 and Runx3 (30-32). The most prominent DNA-binding partners in transcriptional activation In normal mammalian cells and tumor cells without processes, however, are members of the Sp1/KLF-like family alterations of the Smad signaling pathway, the heteromeric of zinc finger proteins, helix-loop-helix leucine zipper R-Smad/Co-Smad complex translocates into the nucleus to transcription factors and members of the Wnt/-catenin/T-cell regulate gene expression (10, 14). Here, the complex can factor (TCF) pathway (10). TFE3, for instance, is a basic either directly bind to DNA or interact with other helix-loop-helix leucine zipper protein that binds to the E box transcription factors to regulate the transcription of TGFβ (CACGTG) and, together with Smad3, induces transcription target genes. Using a bound-oligonucleotide selection from the Smad7 promoter in response to TGFβ. Like TFE3, strategy, the binding specificity of recombinant Smad Sp1, the founder of the Sp/KLF-like family of zinc-finger proteins was originally defined as 5’-GTCTAGAC-3’ and transcription factors, plays a peculiar role in the transcriptional later shown to be 5’-GTCT-3’, or its complement 5’-AGAC- control of inhibitory Smad7 expression and thereby affects 3’, called the Smad-binding element (SBE) (27). However, TGFβ-signaling in cancer (33). We have shown that Sp1 is a since Smad proteins bind DNA with only low affinity, they partner in Smad mediated transcriptional induction of Smad7 must interact with other DNA-binding cofactors to (34). TGFβ-induction causes Smad nuclear translocation and sufficiently bind and regulate target promoter sites. The subsequent binding to DNA-prebound Sp1, resulting in resulting DNA-binding transcriptional complex demands that enhanced Sp1-binding to specific GC-rich promoter boxes the suitable DNA elements for both the Smads and the within the proximal Smad7 promoter. Interestingly, direct partner factor are present at the correct distance and Smad promoter binding is not required in this case, revealing a orientation on the target promoter. Thus, as simply as the novel and previously unknown function of Smad proteins in Smad pathway basically works, the selection of Smad- which they transfer the TGFβ signal from the receptor to the regulated target promoters is not only a matter of Smad target promoter sequence without the need to bind DNA. activation and nuclear translocation, but instead, is a highly Similarily, Smads and Sp1 cooperate in the transcriptional specific and complicated process that requires the cell-type induction of cyclin D1, matrix metalloproteinase MMP-11 and dependent presence and functionality of specific partner calcineurin in response to TGFβ, although the functional transcription factors (2,10,14). In fact, a given cell type that implication and the underlying mechanisms in these target gene does not express a particular Smad-DNA partner will be regulations remains elusive (35).

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Figure 1. TGF β binding to its transmembrane receptor proteins (R1 and R2) initiates the intracellular Smad signalling pathway. Receptor mediated activation of Smad2 and Smad3 induces their accumulation in the nucleus and their association with Smad4. Depending on the cell-type and the given activation status of a cell, a large number of signalling molecules can interact with the Smads or their downstream transcriptional partner proteins, mostly transcriptional activators or repressors, to determine the transcriptional response of a cell to TGFβ stimulation (TF= partnering transcription factor; R1=TGFβ receptor I; R2=TGFβ receptor II; HGF=hepatocyte growth factor; EGF=epidermal growth factor; INFy=Interferon gamma; JAK1=Janus kinase; ERK=extracellular signal-regulated kinase; Stat1=signal transducers and activators of transcription 1; PKC=protein kinase C; CamKII=calmodulin kinase II; JNK=jun N-terminal kinase).

In addition, Smads interact with members of the activating Smad proteins can specifically induce transcription from protein-1 (AP-1) transcription factor family that controls selected TGFβ-target promoters. Most interestingly, however, important aspects of cell proliferation, apoptosis and survival activation of the Smad proteins does not necessarily cause (36). Their transcriptional activities are cell-type dependent target gene induction but instead can result in target promoter and strictly controlled by the differentiation state of the cell suppression upon TGFβ. Microarray-based transcriptome and the type of stimuli it has received. For instance, the analysis has revealed that at least one-quarter of all TGFβ mitogen-activated protein kinase (MAPK) pathways regulate gene responses in mammalian cells are gene repression both its amounts and transactivation capacities (36). In this responses (41-44). The most prominent example is the context, increased activity or expression levels of the tumor transcriptional repression of the c-myc proto-oncogene (41), promoting Smad-partner AP-1 were reported in tumor cells the repression of which is considered the “core” event in with activating mutations of Ras or increased activity of the TGFβ-regulated cell growth inhibition. Rho-GTPase (37). Increased AP-1 activity may thus Repression of inhibitory domain 1 (ID1) is mediated by outbalance competing partner proteins and synergize with Smad complexes with ATF3 and the osteocalcin promoter is Smads in activating AP-1 binding sites of genes involved in regulated through interaction between Smad3 and the tumor cell invasion and metastasis. For instance, AP1 transcription factor Runx2 (42, 45). In summary, TGFβ transcription factors interact with Smads in the regulation of through activation of the Smad signaling pathway controls a oncogenic c-jun and interleukin-11 (38-40). Taken together, large number of different, but specific transcriptional responses. with the help of a large number of DNA-binding proteins, Depending on the cellular context, the presence and activation

1534 Ellenrieder : Sp1/KLF-like Proteins and Smad Gene Regulation Initiated by TGF β ( Review)

Figure 2. The current model of TGFβ-induced cell growth inhibition. TGFβ inhibits cell proliferation mainly through induction of the cell cycle inhibitors p15 and p21, which block G1 cell cycle transition through inactivation of the cyclinD/cdk6 complex. The initial step in the TGFβ cytostatic program, however, is the Smad/E2F4/p130 mediated transcriptional repression of the c-Myc oncogene. An alternative mechanism is mediated by TGFβ-induced nuclear interaction of Smad3 with the Sp1/KLF-like zinc-finger protein KLF11, resulting in c-Myc promoter silencing and cell growth inhibition. In addition, KLF11 increases Smad-mediated signalling and transcription through termination of the negative feedback loop imposed by Smad7 (cdk6=cyclin dependent kinase 6, Rb=retinoblastoma protein).

Figure 3. A) Structural properties of KLF11. The c-terminal domain (CTD) comprises three zinc-finger motifs (ZF) responsible for DNA-binding to GC-rich promoter sequences. The N-terminus (NTD) contains three independent repression domains, called R1, R2 and R3, connected by two linker regions (L1 and L2). The L1 linker region contains four ERK-regulated serine/threonine phosphorylation sites (PLTP, PQSP, PVSP and PSSP). B) Oncogenic Ras-ERK MAPK signalling disrupts KLF11 binding to its corepressor mSin3A and thus blocks KLF11 mediated repression in cancer cells (EGFR=epidermal growth factor receptor; HDAC=histon deacetylase).

1535 ANTICANCER RESEARCH 28 : 1531-1540 (2008) status of DNA-partner transcription factors and co-proteins, The TGFβ-inducible Transcriptional Smads bind to specific promoter elements of TGFβ-regulated Repressors KLF10 and KLF11 target genes and either induce or repress their transcription. The human homolog of KLF10 (TIEG1) was originally The Sp1/KLF-like Family of Transcription Factors identified as the product of a TGFβ-inducible early-response gene from osteoblastic cell populations using differential Sp1 was identified in the early 1980s and was one of the display PCR (52). Raul Urrutia and coworkers subsequently first transcription factors to be purified in mammalian cells reported the cloning of KLF10 (TIEG1) from a rat pancreas (46). It has been shown that Sp1 recognizes and cDNA library and demonstrated its TGFβ-inducibility and specifically binds to GC-rich sites within target promoters functional implication in the regulation of epithelial cell growth through three Cys 2His 2 zinc-finger motifs. A very similar (6). In fact, overexpression of KLF10 mimics TGFβ action in DNA-binding domain had also been found in the several cell types by modulating differentiation markers, Drosophila embryonic pattern regulator Krüppel as well as decreasing proliferation and inducing apoptosis (6, 53-55). in other transcription factors that contain three C 2H2-type Biochemical analysis further revealed that the carboxyl- zinc fingers in their C-terminus highly similar to those of terminal three zinc finger DNA binding domain allows KLF10 Sp1 (47). These proteins are now classified as the family to directly associate with GC-rich sequences of DNA in target of Sp1-like proteins or Krüppel-like factors (KLFs) (48). gene promoters (6, 52, 53). The three zinc fingers are highly In addition to DNA binding, the highly conserved zinc- conserved between KLF10 and the related transcription factor finger motifs may also function in protein-protein KLF11 (TIEG2), suggesting that both factors may regulate the interactions that modulate DNA-binding specificity (49). same sequences in the target gene promoter (53). On the other The amino-terminal regions of the Sp1/KLF-like proteins hand, the N-terminal transcriptional regulatory domain is less are much more variable and contain transcriptional homologous between KLF10 and KLF11, yet both factors have activation or repression domains. In addition, Sp1/KLF-like been shown to function as transcriptional repressors (49, 56). proteins have nuclear localization sequences, which can The N-terminal domain contains three independent occur immediately adjacent to, or within, the zinc-finger transcriptional repression domains called R1, R2, and R3. The motifs (50). Within the Sp1/KLF-like family, several R1 and R2 domains in KLF11 encompass a region of 10-and subgroups have been defined on the basis of sequence and 12-amino acids, respectively, while R3 is a longer proline-rich functional similarities. Sp1–Sp4 form a subgroup which sequence of approximately 80aa (56). Detailed mutation contains several distinct overlapping features/regions which analysis has revealed the presence of a central 10-amino-acid include activation domains (AD), the C-terminal zinc core sequence that is required for the repressor function of R1 finger DNA-binding region and an inhibitory domain in (56). This core sequence has been characterized as the α- Sp3 that is involved in the suppressive activity of Sp3. To helical repression motif SID (Sin3-interacting domain) date, at least 15 KLF proteins have been characterised and responsible for the physical interaction with the Sin3A grouped into different subgroups based on functional and corepressor (57). Interestingly, the SID motif is also a structural specifications (48-50). The KLF-subfamily characteristic feature of the Sp1/KLF-like proteins KLF9, includes the basal transcription element binding (BTEB) KLF13 and KLF16, thus linking the function of these proteins proteins and the TGF β- inducible early response genes to histon deacetylase (HDAC)-mediated transcriptional TIEG1 and TIEG2 (KLF10 and KLF11). The function of repression through mSin3A binding (58). The interaction with individual Sp/KLF-like proteins is continually being mSin3A is of paramount importance for KLF11 mediated gene defined and depends, in part, on the temporal and tissue- silencing and tumor suppression. It has been shown, for specific patterns of individual gene expression. There is instance, that KLF11 inhibits cell growth in vivo when targeted emerging evidence that Sp1/KLF-like proteins are critical to epithelial cells in transgenic mice (8). The growth inhibitory factors in cell growth control and differentiation and that function of KLF11 involves the down-regulation of genes cancer development is often associated with changes of the encoding oxidative stress scavengers, such as SOD2 and expression or function of individual family members. In Catalase1 and requires the presence of an intact SID. fact, some proteins of the family exhibit strong oncogenic functions, while others inhibit tumor outgrowth through The Role of KLF11 in Smad Signalling and transcriptional regulation of selected target genes. The roles Transcription in Normal and Malignant Epithelial of Sp1-proteins in tumor development and metastasis have Cells recently been summarized in several excellent reviews (48, 51). Here, the growth inhibitory function of the TGFβ- The tumor suppressor activities of TGFβ are particularly regulated TIEG proteins (KLF10 and KLF11) and their mediated by its ability to suppress cell growth. The initial roles in TGFβ-regulated transcription, are focused upon. event in TGFβ-induced cell cycle arrest is the rapid down-

1536 Ellenrieder : Sp1/KLF-like Proteins and Smad Gene Regulation Initiated by TGF β ( Review) regulation of c-myc. Promoter analysis has identified a TGFβ- intracellular signalling and transcription pathways including inhibitory element, TIE, within the proximal c-myc promoter, the Smads and the Sp1/KLF-like zinc-finger proteins KLF10 which is required for TGFβ-induced transcriptional down- and KLF11. The transcriptional response of a cell to TGFβ, regulation in epithelial cells. The c-myc TIE is bound by a however, is tightly controlled not only by its downstream complex consisting of Smad3, Smad4, E2F4/DP1 and the Smads and KLF effector proteins, but also by the cell-type corepressor p107 (41, 59). An alternative mechanism is dependent composition of interacting signalling and mediated by promoter bound complex formation of receptor- transcription molecules. This includes crosstalk interactions activated Smad3 and KLF11 (60). Upon TGFβ stimulation, with mitogenic and antiproliferative pathway members as KLF11 interacts with nuclear Smad3 and increases its affinity well as the assembly of specific nuclear transcriptional to bind the TIE element. Disruption of the KLF11 binding complexes. element within the TIE element disables sufficient Smad3 promoter binding and prevents TGFβ-induced repression of c- References myc in epithelial cells. This alternative mechanism is interesting as it identifies a self-enabling mechanism whereby 1 de Caestecker MP, Piek E and Roberts AB: Role of transforming Smad induces expression of its partner protein which then growth factor-beta signaling in cancer. J Natl Cancer Inst 92 : enables another Smad-dependent gene response. A similar 1388-1402, 2000. mechanism has been described in TGFβ-induced repression 2 Moustakas A, Souchelnytskyi S and Heldin CH: Smad regulation of the ID1 promoter. (42). TGFβ increases ATF3 expression in TGF-beta signal transduction. J Cell Sci 114 : 4359-4369, 2001. in a first-wave response, and then stimulates Smad3 and ATF3 3 Dumont N and Arteaga CL: Transforming growth factor-beta and breast cancer: Tumor promoting effects of transforming growth to cooperatively repress ID1 expression through binding to factor-beta. Breast Cancer Res 2: 125-132, 2000. specific promoter elements. In addition to its effects on the 4 Moustakas A and Heldin CH: Non-Smad TGF-beta signals. J human c-myc promoter, KLF11 increases TGFβ-mediated cell Cell Sci 118 : 3573-3584, 2005. growth inhibition through termination of the negative feedback 5 Cook T, Gebelein B, Mesa K, Mladek A and Urrutia R: loop imposed by inhibitory Smad7. When induced by TGFβ, Molecular cloning and characterization of TIEG2 reveals a new KLF11 binds to GC-rich binding elements within the proximal subfamily of transforming growth factor-beta-inducible Sp1-like Smad7 promoter and terminates its induction through zinc finger-encoding genes involved in the regulation of cell growth. J Biol Chem 273 : 25929 -259 36, 1998. recruitment of the mSin3A corepressor complex. This 6 Tachibana I, Imoto M, Adjei PN, Gores GJ, Subramaniam M, mechanism is of particular interest, as it re-sensitizes epithelial Spelsberg TC and Urrutia R: Overexpression of the TGFbeta- cells to TGFβ and enhances Smad-mediated signalling and regulated zinc finger encoding gene, TIEG, induces apoptosis transcription (7). Figure 2 illustrates the current model of in pancreatic epithelial cells. J Clin Invest 99 : 2365-2374, TGFβ-induced cell growth inhibition including the established 1997. functional implications of KLF11. 7 Buck A, Buchholz M, Wagner M, Adler G, Gress T and Taken together, the induction of early response genes Ellenrieder V: The tumor suppressor KLF11 mediates a novel mechanism in transforming growth factor beta-induced growth such as KLF11 is an important step in TGFβ-regulated gene inhibition that is inactivated in pancreatic cancer. Mol Cancer silencing during cell growth inhibition. However, this Res 4: 861-872, 2006. central aspect in the cytostatic program of TGFβ is 8 Fernandez-Zapico ME, Mladek A, Ellenrieder V, Folch-Puy E, fundamentally disturbed during carcinogenesis as shown by Miller L and Urrutia R: An mSin3A interaction domain links the loss of KLF11 repression activity in pancreatic cancer transcriptional activity of KLF11 with its role in growth harbouring K-Ras mutations (7, 61). In fact, we have regulation. EMBO J 22 : 4748-4758, 2003. recently shown that in pancreatic cancer cells with K-Ras 9 Rahimi RA and Leof EB: TGF-beta signaling: a tale of two mutations, activation of ERK MAPK causes responses. J Cell Biochem 102 : 593-608, 2007. 10 Massague J and Gomis RR: The logic of TGFbeta signaling. phosphorylation of the linker region between the repressor FEBS Lett 580 : 2811-2820, 2006. domains R1 and R2 of KLF11, and thus disrupts its ability 11 Tsukazaki T, Chiang TA, Davison AF, Attisano L and Wrana JL: to bind the mSin3A corepressor complex. Consequently, SARA, a FYVE domain protein that recruits Smad2 to the KLF11 loses its ability to terminate transcription of the TGFbeta receptor. Cell 95 : 779-791, 1998. inhibitory Smad7 and to silence the c-Myc oncogene in 12 Xu L, Kang Y, Col S and Massague J: Smad2 nucleocytoplasmic respone to TGFβ (7, 61). Figure 3 illustrates the structural shuttling by nucleoporins CAN/Nup214 and Nup153 feeds properties of KLF11 including the ERK phosphorylation TGFbeta signaling complexes in the cytoplasm and nucleus. Mol Cell 10 : 271-282, 2002. sites and depicts the current model of KLF11 inactivation in 13 Kretzschmar M, Doody J, Timokhina I and Massague J: A cancer cells harbouring oncogenic K- ras mutations. mechanism of repression of TGFbeta/ Smad signaling by In summary, TGFβ controls a plethora a cellular functions oncogenic Ras. Genes Dev 13 : 804-816, 1999. through its ability to regulate gene expression. Target gene 14 Massague J and Wotton D: Transcriptional control by the TGF- selection and regulation occurs through activation of distinct beta/Smad signaling system. EMBO J 19: 1745-54, 2000.

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1538 Ellenrieder : Sp1/KLF-like Proteins and Smad Gene Regulation Initiated by TGF β ( Review)

46 Berg JM: Sp1 and the subfamily of zinc finger proteins with 57 Zhang JS, Moncrieffe MC, Kaczynski J, Ellenrieder V, guanine-rich binding sites. Proc Natl Acad Sci USA 89 : 11109- Prendergast FG and Urrutia R: A conserved alpha-helical motif 11110, 1992. mediates the interaction of Sp1-like transcriptional repressors 47 Turner J and Crossley M: Mammalian Kruppel-like transcription with the corepressor mSin3A. Mol Cell Biol 21 : 5041-5049, factors: more than just a pretty finger. Trends Biochem Sci 24 : 2001. 236-240, 1999. 58 Kaczynski J, Zhang JS, Ellenrieder V, Conley A, Duenes T, 48 Black AR, Black JD, and Azizkhan-Clifford J: Sp1 and kruppel- Kester H, van Der Burg B and Urrutia R: The Sp1-like protein like factor family of transcription factors in cell growth BTEB3 inhibits transcription via the basic transcription element regulation and cancer. J Cell Physiol 188 : 143-160, 2001. box by interacting with mSin3A and HDAC-1 co-repressors and 49 Kaczynski J, Cook T and Urrutia R: Sp1- and Kruppel-like competing with Sp1. J Biol Chem 276 : 36749-36756, 2001. transcription factors. Genome Biol 4: 206, 2003. 59 Frederick JP, Liberati NT, Waddell DS, Shi Y and Wang XF: 50 Lomberk G and Urrutia R: The family feud: turning off Sp1 by Transforming growth factor beta-mediated transcriptional Sp1-like KLF proteins. Biochem J 392 : 1-11, 2005. repression of c-myc is dependent on direct binding of Smad3 to 51 Safe S and Abdelrahim M: Sp transcription factor family and its a novel repressive Smad binding element. Mol Cell Biol 24 : role in cancer. Eur J Cancer 41 : 2438-2448, 2005. 2546-2559, 2004. 52 Subramaniam M, Harris SA, Oursler MJ, Rasmussen K, Riggs 60 Ellenrieder V, Buck A, Harth A, Jungert K, Buchholz M, Adler BL and Spelsberg TC: Identification of a novel TGF-beta- G, Urrutia R and Gress TM: KLF11 mediates a critical regulated gene encoding a putative zinc finger protein in human mechanism in TGF-beta signaling that is inactivated by Erk- osteoblasts. Nucleic Acids Res 23 : 4907-4912, 1995. MAPK in pancreatic cancer cells. Gastroenterology 127 : 607- 53 Cook T and Urrutia R: TIEG proteins join the Smads as TGF- 620, 2004. beta-regulated transcription factors that control pancreatic cell 61 Ellenrieder V, Zhang JS, Kaczynski J and Urrutia R: Signaling growth. Am J Physiol Gastrointest Liver Physiol 278 : G513-521, disrupts mSin3A binding to the Mad1-like Sin3-interacting 2000. domain of TIEG2, an Sp1-like repressor. EMBO J 21 : 2451- 54 Hefferan TE, Reinholz GG, Rickard DJ, Johnsen SA, Waters 2460, 2002. KM, Subramaniam M and Spelsberg TC: Overexpression of a nuclear protein, TIEG, mimics transforming growth factor-beta action in human osteoblast cells. J Biol Chem 275 : 20255- 20259, 2000. 55 Johnsen SA, Subramaniam M, Janknecht R and Spelsberg TC: TGFbeta inducible early gene enhances TGFbeta/Smad- dependent transcriptional responses. Oncogene 21 : 5783-5790, 2002. 56 Cook T, Gebelein B, Belal M, Mesa K and Urrutia R: Three conserved transcriptional repressor domains are a defining Received December 14, 2007 feature of the TIEG subfamily of Sp1-like zinc finger proteins. J Revised February 20, 2008 Biol Chem 274 : 29500-29504, 1999. Accepted March 3, 2008

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