INVITED REVIEW
TIEG proteins join the Smads as TGF-beta -regulated transcription factors that control pancreatic cell growth

Tiffany Cook1 and Raul Urrutia1,2,3

1 Gastroenterology Research Unit, 2 Department of Molecular Neurosciences, and 3 Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55901


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
TGF-beta SIGNALING
EXOCRINE PANCREATIC EPITHELIAL...
MEMBRANE-TO-NUCLEUS TGF-beta ...
MODULAR STRUCTURE OF SMAD...
SMAD PROTEINS IN PANCREATIC...
EARLY-RESPONSE GENES FOR TGF-beta ...
MODULAR STRUCTURE OF TIEG...
TIEG PROTEINS INHIBIT...
DIFFERENCES BETWEEN SMAD AND...
ALTERATIONS IN TGF-beta SIGNALING...
REFERENCES

The control of epithelial cell proliferation, differentiation, and apoptosis requires a balance between signaling and transcriptional regulation. Recent developments in pancreatic cell research have revealed that transforming growth factor-beta (TGF-beta ) signaling is important for the regulation of each of these phenomena. More importantly, perturbations in this pathway are associated with pancreatic cancer. A chief example of these alterations is the mutation in the TGF-beta -regulated transcription factor Smad4/DPC4 that is found in a large percentage of pancreatic tumors. Surprisingly, studies on transcription factors have remained an underrepresented area of pancreatic research. However, the discovery of Smad4/DPC4 as a transcription factor fueled further studies aimed at characterizing transcription factors involved in normal and neoplastic pancreatic cell growth. Our laboratory recently described the existence of a novel family of zinc finger transcription factors, TGF-beta -inducible early-response gene (TIEG)1 and TIEG2, from the exocrine pancreas that, similarly to Smads, participate in the TGF-beta response and inhibit epithelial cell proliferation. This review therefore focuses on describing the structure and function of these two families of transcription factor proteins that are becoming key players in the regulation of pancreatic cell growth.

early-response genes; apoptosis; proliferation; differentiation; zinc finger transcription factor


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
TGF-beta SIGNALING
EXOCRINE PANCREATIC EPITHELIAL...
MEMBRANE-TO-NUCLEUS TGF-beta ...
MODULAR STRUCTURE OF SMAD...
SMAD PROTEINS IN PANCREATIC...
EARLY-RESPONSE GENES FOR TGF-beta ...
MODULAR STRUCTURE OF TIEG...
TIEG PROTEINS INHIBIT...
DIFFERENCES BETWEEN SMAD AND...
ALTERATIONS IN TGF-beta SIGNALING...
REFERENCES

OVER THE PAST TWO DECADES, extensive studies have led to the design of a working model to explain how cells regulate proliferation, differentiation, and apoptosis. For this purpose, a cell must transduce extracellular signals and translate them into a program of gene expression. In a number of cell systems, this occurs by extracellular peptide ligands binding to a cell surface receptor to initiate a cytoplasmic signaling cascade. This cascade leads to posttranslational modifications of intracellular proteins that then elicit short-term responses (e.g., cytoskeleton reorganization or endocytosis) or more long-term responses (e.g., cell growth). For long-term responses, the signal emanates from the cytoplasm into the nucleus to regulate transcription factor proteins that activate or repress the expression of genes necessary for cell growth control (Fig. 1). Therefore, homeostasis of cell growth relies on an interplay between cell signaling and transcriptional regulation. This tight control of cellular growth is of paramount importance for organogenesis and regeneration, and alterations in these processes lead to embryonic malformations, healing defects, and neoplastic transformation. One of the best examples of a signaling pathway that regulates cell growth is that mediated by transforming growth factor-beta (TGF-beta ) peptides in epithelial cells, the major topic of discussion of this review. Under normal conditions, TGF-beta peptides inhibit epithelial cell proliferation and induce apoptosis (19, 63). Interestingly, the disruption of this pathway in many organs, including the pancreas, appears to be an important step in the development of cancer (19, 62, 80). Thus the identification and characterization of the molecular machinery underlying the ability of TGF-beta to control epithelial cell growth will have a remarkable impact on human health.


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Fig. 1.   General model for regulation of cell growth. An extracellular ligand (e.g., growth factor, GF) binds to a transmembrane receptor to initiate a cytoplasmic signaling cascade. This cascade emanates into the nucleus where it regulates transcription factors that can turn on or turn off genes necessary for proliferation, differentiation, or apoptosis.


    TGF-beta SIGNALING
TOP
ABSTRACT
INTRODUCTION
TGF-beta SIGNALING
EXOCRINE PANCREATIC EPITHELIAL...
MEMBRANE-TO-NUCLEUS TGF-beta ...
MODULAR STRUCTURE OF SMAD...
SMAD PROTEINS IN PANCREATIC...
EARLY-RESPONSE GENES FOR TGF-beta ...
MODULAR STRUCTURE OF TIEG...
TIEG PROTEINS INHIBIT...
DIFFERENCES BETWEEN SMAD AND...
ALTERATIONS IN TGF-beta SIGNALING...
REFERENCES

TGF-beta is a member of an evolutionarily conserved family of secreted peptides that can modulate cell differentiation, arrest cell proliferation, and/or induce apoptosis in most epithelial cell populations. Structurally, this family of peptides is grouped into three major subfamilies: TGF-beta s, activins, and bone morphogenetic proteins (BMPs) (for recent reviews, see Refs. 19 and 62). During the last decade, the identification of both TGF-beta receptors and TGF-beta -inducible transcription factors has revealed the existence of a direct signaling pathway from the cell membrane to the nucleus. This pathway is responsible for regulating the expression of genes that control cell growth. The medical relevance of this research is underscored by the fact that mutations in either TGF-beta receptors or TGF-beta -inducible transcription factors result in the failure of cells to respond to antiproliferative and/or apoptotic signals and in the development of cancer. This evidence points to the crucial physiological role of these molecules in the control of normal epithelial cell growth, differentiation, and apoptosis. Several excellent reviews have recently been published on the growing field of TGF-beta signaling (1, 19, 62, 63, 80). This review therefore focuses on recent advances in our understanding of the role of two TGF-beta -regulated transcription factor families, the TGF-beta -inducible early-response gene (TIEG) and Smad proteins, in exocrine pancreatic cells.


    EXOCRINE PANCREATIC EPITHELIAL CELLS ARE A USEFUL MODEL FOR STUDYING EFFECTS OF TGF-beta PEPTIDES IN CELL GROWTH CONTROL AND PANCREATIC CANCER
TOP
ABSTRACT
INTRODUCTION
TGF-beta SIGNALING
EXOCRINE PANCREATIC EPITHELIAL...
MEMBRANE-TO-NUCLEUS TGF-beta ...
MODULAR STRUCTURE OF SMAD...
SMAD PROTEINS IN PANCREATIC...
EARLY-RESPONSE GENES FOR TGF-beta ...
MODULAR STRUCTURE OF TIEG...
TIEG PROTEINS INHIBIT...
DIFFERENCES BETWEEN SMAD AND...
ALTERATIONS IN TGF-beta SIGNALING...
REFERENCES

Our laboratory studies cell cycle arrest and apoptosis mediated by TGF-beta -inducible transcription factors in exocrine pancreatic cell populations. The pancreas provides an excellent model for studying TGF-beta -based phenomena, and the system is medically relevant because alterations in this signaling cascade have been associated with the development of pancreatic cancer (5, 17, 26, 27, 61, 82). The adult pancreas contains an endocrine portion comprised of four different cell populations (alpha , beta , gamma , and delta ) and an exocrine portion comprised of acinar and ductular cells. Interestingly, all six of these pancreatic cell populations are believed to originate from an epithelial "ductular-like" precursor, a phenotype that is characteristic of cancerous pancreatic cells (10).

The effects of TGF-beta on pancreatic cell differentiation were originally examined using a whole organ culture system. In this system, TGF-beta has an inhibitory effect on acinar cell growth but also increases the number of islet cells differentiated from ductular-like structures, indicating that TGF-beta may exert differential effects on cell differentiation in a cell-specific manner (30, 73). The central role of TGF-beta in both pancreatic cell differentiation and growth has recently been confirmed and extended in vivo using transgenic animals. For example, overexpression of TGF-beta 1 specifically in the pancreatic beta -cells of transgenic mice not only alters islet cell development but also inhibits acinar cell proliferation (52, 74). Furthermore, transgenic mice that express a dominant-negative TGF-beta receptor type II in the acinar cells of the pancreas (rendering these cells insensitive to TGF-beta signaling) exhibit increased proliferation and perturbed differentiation of acinar cells (9). Together, these results confirm that TGF-beta signaling exerts a negative effect on acinar cell growth and that it is also necessary for maintaining the differentiation status of both acinar and islet cells.

Other useful models for studying the growth-regulatory effects of TGF-beta in pancreas are provided by several transformed cell lines derived from human pancreatic cancers or chemically induced tumors from experimental animals. For example, several of these exocrine pancreatic cell lines are responsive to TGF-beta peptides and can be used for in vitro anchorage-dependent and -independent cell growth assays (5, 20, 33, 41, 43, 45, 46, 51, 55, 72, 83). In addition, because these cells often carry complex genetic alterations, including mutations in several mediators of TGF-beta signaling, the results obtained from using these lines can be informative when dissecting specific defects in this pathway (5, 23, 33, 83, 85). Furthermore, the data described above are in agreement with a previous report by Logsdon et al. (60) that described for the first time the growth-inhibitory effects of TGF-beta on normal isolated pancreatic acinar cells, validating the extrapolation of results obtained in cell lines to physiological conditions. In summary, a large variety of experimental models exist for testing the role of TGF-beta on pancreatic cell growth in vitro and in vivo, namely pancreas explants, isolated cell populations, and transgenic animal models. The theoretical framework emerging from the combined use of these systems points toward an essential role for the TGF-beta pathway in pancreatic organogenesis and tumor suppression. Finally, we are optimistic that many of the concepts derived by using pancreatic cells can be applied to other epithelial cell systems.


    MEMBRANE-TO-NUCLEUS TGF-beta SIGNALING VIA SMAD PROTEINS
TOP
ABSTRACT
INTRODUCTION
TGF-beta SIGNALING
EXOCRINE PANCREATIC EPITHELIAL...
MEMBRANE-TO-NUCLEUS TGF-beta ...
MODULAR STRUCTURE OF SMAD...
SMAD PROTEINS IN PANCREATIC...
EARLY-RESPONSE GENES FOR TGF-beta ...
MODULAR STRUCTURE OF TIEG...
TIEG PROTEINS INHIBIT...
DIFFERENCES BETWEEN SMAD AND...
ALTERATIONS IN TGF-beta SIGNALING...
REFERENCES

The discovery of DPC4 (deleted in pancreatic cancer 4) as a putative tumor suppressor gene for pancreatic cancer in 1996 was a breakthrough in the field of pancreatic cell research (35). The finding that the product of DPC4 was structurally related to Mad, a protein involved in TGF-beta -related signaling in Drosophila, paved the way toward the identification of an entire family of Smad proteins that function as important mediators of TGF-beta signaling pathways (50, 58). Currently, eight different Smad-encoding genes have been isolated from mammalian cell populations (Smad 1-8), and they are classified into three different subfamilies: receptor-regulated Smads (R-Smads), co-Smads, and anti-Smads (see Refs. 4, 49, 62, and 86 for reviews). The R-Smads bind to a specific TGF-beta -related receptor at the cell surface, are phosphorylated at their COOH termini on ligand stimulation, associate with a co-Smad, and translocate into the nucleus to regulate gene expression (Fig. 2A). R-Smads 2 and 3 both participate in TGF-beta and activin signaling, whereas R-Smads 1, 5, and 8 participate in BMP-mediated signaling. In contrast, the only co-Smad identified thus far, Smad4/DPC4, associates with Smads 1-3 and 5 and thus participates in TGF-beta , activin, and BMP signaling. Finally, Smad6 and Smad7 function as anti-Smads by interfering with R-Smad function. This interference occurs either by binding to the TGF-beta receptor, blocking phosphorylation of R-Smads (Smad6), or by binding directly to an R-Smad to prevent interaction with Smad4 (Smad7).


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Fig. 2.   Summary of Smad protein function and structure. A: under noninduced conditions, receptor-regulated Smads (R-Smads) associate with transforming growth factor-beta (TGF-beta ) receptors (Tbeta Rs) at cell surface. On receptor activation, R-Smads become phosphorylated by its cognate receptor at a COOH-terminal SSXS site, dissociate from the receptor, and form a complex with a co-Smad. This complex then translocates into the nucleus to regulate gene expression that leads to the cellular effects of TGF-beta signaling. B: R-Smads, co-Smads, and anti-Smads share a similar structure of Mad homology (MH)1, linker, and MH2 domains (described in text). In addition, R-Smads contain a receptor phosphorylation site, SXSS, at the COOH terminus and co-Smads contain an insert sequence of ~30 amino acids within the MH2 domain. Anti-Smads have a poorly conserved MH1 domain (hatched bars) and thus do not bind DNA. BMP, bone morphogenetic protein.


    MODULAR STRUCTURE OF SMAD PROTEINS
TOP
ABSTRACT
INTRODUCTION
TGF-beta SIGNALING
EXOCRINE PANCREATIC EPITHELIAL...
MEMBRANE-TO-NUCLEUS TGF-beta ...
MODULAR STRUCTURE OF SMAD...
SMAD PROTEINS IN PANCREATIC...
EARLY-RESPONSE GENES FOR TGF-beta ...
MODULAR STRUCTURE OF TIEG...
TIEG PROTEINS INHIBIT...
DIFFERENCES BETWEEN SMAD AND...
ALTERATIONS IN TGF-beta SIGNALING...
REFERENCES

The Smad proteins share a general structure containing two highly conserved domains, most commonly referred to as Mad homology domains 1 and 2 (MH1 and MH2), separated by a variable proline-rich linker region (Fig. 2B). Within the R-Smads and co-Smads, the MH1 domain, located at the NH2 terminus, is a 130-amino acid sequence that serves as a unique DNA binding motif. The anti-Smads, however, have limited homology within this domain, and consequently have not been shown to behave as DNA binding proteins. DNA binding activity of Smad proteins was first observed in Drosophila melanogaster, in which Kim et al. (44) demonstrated that the MH1 domain of Mad, when fused to the heterologous Gal4 transactivation domain, could regulate a number of target genes of the TGF-beta -like peptide Dpp. Since this finding, several studies have begun to define a consensus binding site for Smad proteins in mammalian cells. Experiments using endogenous target gene promoters as well as random oligonucleotide binding assays have demonstrated that the consensus binding site for Smads contains a core AGAC sequence (18, 21, 59, 84, 90). Interestingly, however, TGF-beta -like response genes often require the presence of not only Smad binding sites but also binding sites for other transcription factors to achieve appropriate expression. The activin-inducible gene, Mix.2, for example, requires the interaction between Smad2 and the DNA binding protein forkhead activin signal transducer-1 (FAST-1) to regulate its expression (11). FAST-1 is a hepatocyte nuclear factor-beta (HNF-beta )/forkhead-related transcription factor that was shown to bind to Smad2 in a yeast two-hybrid screen (12). Subsequent studies demonstrated that Smad2/Smad4 or FAST-1 alone is unable to regulate Mix.2 expression but together drive activin-dependent activation. Furthermore, binding of FAST-1 to a site located directly upstream of the Smad2 binding site is necessary for this regulation (12, 59). Together, these data suggest that DNA binding specificity of the Smad proteins is facilitated by cooperative binding with other transcription factors. This hypothesis has been further supported by recent reports demonstrating cooperative transcriptional regulation of gene expression through interaction with the transcription factors AP-1, Sp1, transcription factor µE3, and the vitamin D receptor (VDR) (37, 66, 89, 92). These studies lend insight into the mechanisms underlying promoter recognition by the Smad proteins, a crucial step toward defining in vivo targets of TGF-beta signaling.

The MH2 domain, located at the COOH terminus of the Smad proteins, is highly conserved among all family members and is important for several functional processes, including homo- and heterooligomerization, receptor binding, nuclear localization, phosphorylation, and transcriptional regulation (36, 62, 86). Under resting conditions, the MH2 domain is important for association of the R-Smads to their cognate receptors as well as homotrimerization of the Smad molecules, an association that appears to maintain them in an inactive state. On ligand stimulation, a SSXS motif at the very COOH terminus of the R-Smads is phosphorylated by its receptor, allowing heterodimerization between the R-Smads and the co-Smads and translocation into the nucleus. Once in the nucleus, the R-Smad/co-Smad complex binds to DNA, where it can either activate or repress transcription. The ability of Smads to behave as activators or repressors appears to depend on the interaction of the MH2 domain of these proteins with cofactors that either affect chromatin structure or interact with the basal transcriptional apparatus. For example, CBP/p300, a histone acetyltransferase oncoprotein that loosens nucleosome structure, interacts with various Smads to facilitate transcriptional initiation (22, 39, 67, 70, 76, 81). Conversely, another Smad2-interacting cofactor, TG sequence-interacting factor (TGIF), is a homeodomain protein that recruits a histone deacetylase complex to repress transcription (87). This finding is important because TGF-beta signaling involves not only the activation of gene expression but also transcriptional repression (62). Therefore, through the selective interaction with histone acetylases and deacetylases, Smads are capable of increasing or decreasing gene expression, respectively, underscoring the importance of Smad-mediated transcriptional regulation in TGF-beta signaling.

Finally, the linker domain of Smad proteins contains consensus tyrosine phosphorylation sites that are targets of various signaling cascades that regulate their functions. For instance, sites within the linker regions of Smad1, 2, and 3 are phosphorylated by mitogen-activated protein (MAP) kinase in response to epidermal growth factor (EGF), resulting in their inactivation (47, 48). Similarly, an oncogenic form of Ras interferes with the function of Smad2 and Smad3 through MAP kinase-mediated phosphorylation (48). Thus inactivation of Smads by linker phosphorylation can be used by EGF to antagonize the antiproliferative signals from TGF-beta . Because the linker region varies significantly in structure among the Smads, different phosphorylation sites may provide a mechanism for distinct signaling cascades to differentially regulate the Smad proteins.


    SMAD PROTEINS IN PANCREATIC CELL GROWTH
TOP
ABSTRACT
INTRODUCTION
TGF-beta SIGNALING
EXOCRINE PANCREATIC EPITHELIAL...
MEMBRANE-TO-NUCLEUS TGF-beta ...
MODULAR STRUCTURE OF SMAD...
SMAD PROTEINS IN PANCREATIC...
EARLY-RESPONSE GENES FOR TGF-beta ...
MODULAR STRUCTURE OF TIEG...
TIEG PROTEINS INHIBIT...
DIFFERENCES BETWEEN SMAD AND...
ALTERATIONS IN TGF-beta SIGNALING...
REFERENCES

Although several of the experiments described above have been performed in other ductular epithelial cells such as breast cancer cells, emerging evidence indicates that Smad-mediated processes are fundamental for maintaining the homeostasis of pancreatic cells. The finding that DPC4/Smad4 is mutated in ~50% of pancreatic cancers suggested its participation in a signaling pathway that suppresses neoplastic cell growth in this gland (35). This hypothesis has been further supported by the finding that TGF-beta requires DPC4 to inhibit pancreatic cell growth through the upregulation of the cell cycle inhibitor protein p21waf1 (33). Furthermore, recent studies have revealed the role of other members of the Smad family in the regulation of TGF-beta signaling and pancreatic cell growth. For instance, the anti-Smad protein, Smad6, has been found to antagonize the growth-inhibitory effects of TGF-beta in pancreatic carcinoma cell lines and is overexpressed in pancreatic tumors (45). This mechanism may contribute to the resistance to TGF-beta signaling that is frequently observed in neoplastic pancreatic cell populations. Together, these studies indicate that both mutations in Smads involved in mediating the TGF-beta response and overexpression of anti-Smads are associated with uncontrolled pancreatic cell growth. Thus studies aimed at experimentally modifying these proteins may be useful for devising therapeutic agents for neoplastic diseases of the pancreas.


    EARLY-RESPONSE GENES FOR TGF-beta SIGNALING: THE TIEG PROTEINS
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ABSTRACT
INTRODUCTION
TGF-beta SIGNALING
EXOCRINE PANCREATIC EPITHELIAL...
MEMBRANE-TO-NUCLEUS TGF-beta ...
MODULAR STRUCTURE OF SMAD...
SMAD PROTEINS IN PANCREATIC...
EARLY-RESPONSE GENES FOR TGF-beta ...
MODULAR STRUCTURE OF TIEG...
TIEG PROTEINS INHIBIT...
DIFFERENCES BETWEEN SMAD AND...
ALTERATIONS IN TGF-beta SIGNALING...
REFERENCES

The activation of transcription factors is an absolute requirement for eliciting long-term cellular responses to an extracellular signaling cascade. This mechanism in turn switches on and off the expression of genes that are necessary for a particular cellular event such as proliferation, differentiation, or apoptosis. The first genes to be turned on by a signaling cascade are called "early-response genes." Because of the immediate need for these gene products to respond to an extracellular stimulus, the cellular machinery necessary for their expression is constitutively present in the cell. Thus activation of early-response genes occurs in the absence of de novo protein synthesis. Many of the early-response genes that have been identified for TGF-beta signaling are oncogenic transcription factors (e.g., c-fos, c-jun, and c-myc), pointing to the importance of early-response genes in maintaining normal cell growth (54, 69, 78, 91). As a result of our interest in the regulation of pancreatic epithelial cell growth and neoplastic transformation, we recently revealed the existence of a novel family of pancreas-enriched transcription factors, TIEG1 and TIEG2, that are early-response genes for TGF-beta and inhibit epithelial cell proliferation (14, 79) (Fig. 3).


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Fig. 3.   A: TGF-beta -inducible early-response gene (TIEG) function. TGF-beta signaling immediately upregulates TIEG1 and TIEG2 expression. These early-response transcription factors then regulate genes necessary to inhibit epithelial cell proliferation. Thus overexpression of these gene products is sufficient to induce TGF-beta -mediated responses in these cells. B: TIEG protein structure. TIEG1 and TIEG2 share an overall structural homology of a 44% similar proline-rich NH2 terminus and 3 Sp1-like zinc finger motifs at the COOH terminus that share >91% similarity. Biochemical analysis of the TIEG proteins indicates presence of 3 highly conserved transcriptional repression domains, R1, R2, and R3, within the NH2 terminus that behave as potent transcriptional repression domains (gray bars). In addition, zinc finger region is responsible for binding to GC-rich DNA binding sites and, at least in the case of TIEG2, allows nuclear localization.

The human homolog of TIEG1 was originally identified as the product of a TGF-beta -inducible early-response gene from osteoblastic cell populations using differential display PCR (77). Our laboratory subsequently reported the cloning of TIEG1 from a rat pancreas cDNA library (79). Interestingly, TIEG1 has also been identified as a growth factor-inducible gene from prostate and brain, where it has received the names early growth response gene-alpha (EGR-alpha ) and glial cell-derived neurotrophic factor inducible transcription factor (GIF), respectively (8, 88). These data support a role for TIEG1 in the regulation of growth in many cell systems. In addition, we have recently reported the identification of TIEG2, a protein structurally related to TIEG1 (14). These two proteins are 91% similar within the COOH-terminal DNA binding domain and 44% similar throughout a proline-rich NH2-terminal domain (Fig. 3). Like TIEG1, TIEG2 is also an early-response gene for TGF-beta in pancreatic cell populations (14). Thus on the basis of their sequence similarity and TGF-beta inducibility, TIEG1 and TIEG2 represent a distinct group of transcription factors.


    MODULAR STRUCTURE OF TIEG PROTEINS
TOP
ABSTRACT
INTRODUCTION
TGF-beta SIGNALING
EXOCRINE PANCREATIC EPITHELIAL...
MEMBRANE-TO-NUCLEUS TGF-beta ...
MODULAR STRUCTURE OF SMAD...
SMAD PROTEINS IN PANCREATIC...
EARLY-RESPONSE GENES FOR TGF-beta ...
MODULAR STRUCTURE OF TIEG...
TIEG PROTEINS INHIBIT...
DIFFERENCES BETWEEN SMAD AND...
ALTERATIONS IN TGF-beta SIGNALING...
REFERENCES

With the goal of understanding better the function of TIEG1 and TIEG2, we characterized the biochemical properties of these proteins. One of the most striking features of these proteins is that they each possess three structurally related zinc finger DNA binding motifs at the COOH terminus that share strong homology with members of the Sp1-like family of zinc finger transcription factors. The founding member of this family, Sp1, uses this motif to bind to GC-rich sequences (40). Interestingly, these GC-rich sequences contribute to the regulation of a large number of growth-regulatory genes, including cell cycle regulators (p15, p21, p27), MAP kinases (MAPK), mitogenic GTPases (H-ras), DNA synthesis proteins [histones, thymidine kinase, dihydrofolate reductase (DHFR), DNA polymerase Delta  (POLD1)], growth factors [platelet-derived growth factor (PDGF), TGF-beta , fibroblast growth factor (FGF), granulocyte macrophage colony-stimulating factor (GMCSF), tumor necrosis factor-alpha (TNF-alpha )], and growth factor receptors (insulin receptor and insulin-like growth factor receptor) (2, 3, 6, 7, 29, 38, 42, 53, 56, 57, 64, 65, 68, 71, 93, 94). Therefore, it is not surprising to find that many of the Sp1-like family members function as key regulators of morphogenesis (reviewed in Ref. 15). As predicted from structural analysis, biochemical studies of TIEG1 and TIEG2 demonstrate that both of these proteins recognize Sp1-related GC-rich sequences (14, 16, 88). Of particular interest, however, is that although the majority of Sp1-related proteins function to activate gene expression, both TIEG proteins function as potent transcriptional repressors (13, 15, 88). This repression activity resides within the region of these proteins located NH2-terminal to the zinc finger DNA binding motif. Furthermore, mutational analysis of the NH2 terminus has revealed the presence of three conserved motifs that function as strong repressors (13). In addition, a basic tetrapeptide within the second zinc finger of TIEG2 functions as a nuclear localization signal (NLS) (Ref. 28 and unpublished data). Finally, four linker regions separate the transcriptional repressor and DNA binding motifs. Although the role of these linker regions currently remains undefined, they contain numerous sites for posttranslational modifications that may be targets for different signaling pathways (V. Ellenreider and R. Urrutia, unpublished data). Thus, like the Smads, TIEG transcription factors are modular proteins containing a DNA binding motif, an NLS, a transcriptional regulatory domain, and linker regions that contain sites for posttranslational modifications. Together, these data support a model in which TGF-beta binds to its cell surface receptor to induce the expression of TIEG1 and TIEG2, which in turn bind to GC-rich sequences to repress genes necessary for inhibiting epithelial cell growth.


    TIEG PROTEINS INHIBIT EPITHELIAL CELL GROWTH
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ABSTRACT
INTRODUCTION
TGF-beta SIGNALING
EXOCRINE PANCREATIC EPITHELIAL...
MEMBRANE-TO-NUCLEUS TGF-beta ...
MODULAR STRUCTURE OF SMAD...
SMAD PROTEINS IN PANCREATIC...
EARLY-RESPONSE GENES FOR TGF-beta ...
MODULAR STRUCTURE OF TIEG...
TIEG PROTEINS INHIBIT...
DIFFERENCES BETWEEN SMAD AND...
ALTERATIONS IN TGF-beta SIGNALING...
REFERENCES

The molecular and biochemical characterization of TIEG proteins described above supports a role for these transcription factors as links between TGF-beta signaling cascades and the regulation of nuclear events. To test this role, we characterized the growth-regulatory properties of these proteins both in vitro and in vivo. In vitro studies of TIEG1 revealed that overexpression of this gene in the TGF-beta -sensitive exocrine pancreatic cell line PANC-1 inhibits cell proliferation and also induces apoptosis (Fig. 4, A-C; Ref. 79). These results demonstrate that the increased intracellular levels of TIEG1 achieved using transient transfection mimic the antiproliferative and apoptotic effects of TGF-beta on epithelial cell growth, suggesting that TIEG1 is an important factor for mediating TGF-beta signaling. Similar studies performed with TIEG2 indicate that this TGF-beta -inducible transcription factor is also sufficient to inhibit epithelial cell proliferation (14). Together, the TGF-beta inducibility and the growth-inhibitory properties of the TIEG proteins suggest that these transcription factors may function as signal transducer molecules for TGF-beta -regulated cell growth.


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Fig. 4.   Induction of apoptosis by TIEG proteins. A-C: human pancreatic epithelial cell line PANC-1 was transfected with TIEG1 (A, red), and TIEG1-positive cells were analyzed for apoptosis using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling technique (TUNEL) (B, green). Other morphological changes characteristic of apoptosis, such as chromatin condensation and cytoplasmic disorganization, were monitored by differential interference contrast microscopy (C). Nontransfected cells in same field and cells transfected with vector alone were negative for apoptosis (A-C; Ref. 79). D-F: thin sections of pancreata isolated from negative (D) or positive (E) TIEG2 transgenic animals were analyzed for apoptosis using TUNEL technique as in B. A 10-fold increase in apoptosis is observed in TIEG2-positive vs. -negative animals (A. Mladek and R. Urrutia, unpublished data). Hallmarks of apoptosis were also observed in TIEG2 transgenic pancreata by electron microscopy, including condensation of DNA and organelles, as well as cellular fragmentation. Panels A-C are reproduced with permission from Intl. J. Pancreatol. 22: 1-14, 1997; copyright 1997, Humana Press.

To better understand the role of TIEG proteins in vivo, we have recently used the elastase I promoter to generate transgenic mice that express TIEG2 in the acinar cells of the exocrine pancreas (65). Morphological evaluation of the pancreas at 12 wk revealed a remarkable degree of acinar cell pleomorphism, an increase in the nucleus-to-cytoplasm ratio, and incipient loss of the acini-like organization (Fig. 4, D-F). Interestingly, these changes are reminiscent of the early stages of pancreatic atrophy previously described in mice overexpressing TGF-beta 1. Furthermore, TIEG2 transgenic pancreata show an increase in the rate of apoptosis, one of the expected outcomes of expression of a protein that works in the TGF-beta pathway to control pancreatic cell growth (A. Mladek and R. Urrutia, unpublished data). These results demonstrate that normal levels of the TGF-beta -inducible transcription factor TIEG2 are necessary for maintenance of the homeostasis of pancreatic acinar cells and that perturbations in this pathway can result in significant alterations in pancreatic morphogenesis.


    DIFFERENCES BETWEEN SMAD AND TIEG PROTEINS
TOP
ABSTRACT
INTRODUCTION
TGF-beta SIGNALING
EXOCRINE PANCREATIC EPITHELIAL...
MEMBRANE-TO-NUCLEUS TGF-beta ...
MODULAR STRUCTURE OF SMAD...
SMAD PROTEINS IN PANCREATIC...
EARLY-RESPONSE GENES FOR TGF-beta ...
MODULAR STRUCTURE OF TIEG...
TIEG PROTEINS INHIBIT...
DIFFERENCES BETWEEN SMAD AND...
ALTERATIONS IN TGF-beta SIGNALING...
REFERENCES

The preceding paragraphs mainly stress the similarities between Smads and TIEG proteins that have been described thus far. However, some important differences between these two families of proteins must be considered. For instance, Smads interact directly with the TGF-beta receptor at the cytosolic surface of the cell membrane and move into the nucleus on activation of this signaling cascade. Whether TIEG proteins behave in a similar fashion remains to be established. On the basis of the pattern of expression of TIEG1 and TIEG2, however, these transcription factors appear to be downstream of the Smad proteins. It should also be mentioned, however, that by immunohistochemistry, TIEG1 has been found in both the cytoplasm and the nucleus of exocrine pancreatic cells. (79). Thus it remains a possibility that, similar to the Smads, TIEG proteins undergo nuclear translocation in response to distinct signaling cascades.

In addition to differences between Smad and TIEG proteins, it is also likely that future studies will uncover distinct roles for each of the TIEG proteins. Currently, no differences in the transcriptional regulatory or DNA binding properties have been observed (Ref. 13; T. Cook, A. Mladek, and R. Urrutia, unpublished data). However, it is unlikely that these two proteins exist within a single cell population to perform the same function. Therefore, ongoing studies aimed at determining the exact function of TIEG1 and TIEG2 in vivo will be critical for understanding the participation of each of these proteins in the regulation of pancreatic cell growth.


    ALTERATIONS IN TGF-beta SIGNALING IN PANCREATIC DISEASE
TOP
ABSTRACT
INTRODUCTION
TGF-beta SIGNALING
EXOCRINE PANCREATIC EPITHELIAL...
MEMBRANE-TO-NUCLEUS TGF-beta ...
MODULAR STRUCTURE OF SMAD...
SMAD PROTEINS IN PANCREATIC...
EARLY-RESPONSE GENES FOR TGF-beta ...
MODULAR STRUCTURE OF TIEG...
TIEG PROTEINS INHIBIT...
DIFFERENCES BETWEEN SMAD AND...
ALTERATIONS IN TGF-beta SIGNALING...
REFERENCES

As described in this review, diverse experimental approaches reveal that the normal functioning of TGF-beta signaling is essential for the homeostasis of pancreatic cell physiology. Because of the importance of TGF-beta signaling in the regulation of normal pancreatic cell growth and differentiation, it can be predicted that alterations in these pathways will result in pancreatic diseases. Indeed, several studies have suggested that many, if not all, pancreatic tumor cells exhibit nonfunctional TGF-beta signaling (5, 46, 61, 83). Furthermore, alterations in many components within the TGF-beta signaling pathway, including TGF-beta ligands, receptors, Smads, and early-response transcription factors, have been associated with pancreatitis and pancreatic cancer (5, 23-27, 31, 32, 34, 45, 61, 75, 82, 83, 85). Here, we have reviewed the important roles of two families of transcription factor proteins, the Smads and TIEGs, as critical mediators of the TGF-beta response of pancreatic cells. In addition, we have described the most recent and fundamental studies devoted to the molecular, biochemical, and functional characterization of these proteins. Although these studies constitute an exciting beginning, much more remains to be learned about the role of these proteins in pancreatic cell physiology and disease. This endeavor will certainly involve the combined efforts of several laboratories around the world and will provide the theoretical framework for a new generation of investigators dedicated to understanding the integration of signaling cascades and gene expression. The translation of this basic knowledge into understanding of the mechanisms of pancreatic disease, however, currently constitutes one of the most challenging efforts facing molecular medicine today.


    ACKNOWLEDGEMENTS

The authors thank Brian Gebelein, Karen Hedin, and Vijay Shaw for critically reviewing the manuscript.


    FOOTNOTES

This work was supported by the Mayo Cancer Center and by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant DK-52913 to R. Urrutia. T. Cook was supported by NIDDK Training Grant DK-07198.

Current address for T. Cook: 100 Washington Square E., 1009 Main Building, New York Univ., New York, NY 10003.

Address for reprint requests and other correspondence: R. Urrutia, Gastroenterology Research Unit, 2-445A Alfred Bldg., Mayo Clinic, 200 First St. SW, Rochester, MN 55905 (E-mail: urrutia.raul{at}mayo.edu).


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
TGF-beta SIGNALING
EXOCRINE PANCREATIC EPITHELIAL...
MEMBRANE-TO-NUCLEUS TGF-beta ...
MODULAR STRUCTURE OF SMAD...
SMAD PROTEINS IN PANCREATIC...
EARLY-RESPONSE GENES FOR TGF-beta ...
MODULAR STRUCTURE OF TIEG...
TIEG PROTEINS INHIBIT...
DIFFERENCES BETWEEN SMAD AND...
ALTERATIONS IN TGF-beta SIGNALING...
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