Dynamic Transcriptional Changes of TIEG1 and TIEG2 During Mouse Tissue Development.код для вставкиСкачать
THE ANATOMICAL RECORD 293:858?864 (2010) Dynamic Transcriptional Changes of TIEG1 and TIEG2 During Mouse Tissue Development LEI JIANG,1,2 YANGCHAO CHEN,3 CHU-YAN CHAN,2 GANG LU,4 HUA WANG,2 JI-CHENG LI,1* AND HSIANG-FU KUNG2* 1 Institute of Cell Biology, Zhejiang University, Hangzhou, People?s Republic of China 2 Stanley Ho Centre for Emerging Infectious Diseases, The Chinese University of Hong Kong, Shatin, HKSAR 3 Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Shatin, HKSAR 4 Department of Surgery, The Chinese University of Hong Kong, Shatin, HKSAR ABSTRACT TGF-b-inducible early-response gene (TIEG) is a family of primary response genes induced by TGF-b, which are well recognized in regulating cellular proliferation and apoptosis. However, their expression pro?le has never been investigated during embryogenesis in different organs. In this study, we aimed to investigate the transcriptional level of both TIEG1 and TIEG2 during development in various mice organs, including the brain cortex, cerebellum and stem, brain striatum, muscle, heart, liver, kidney, and lung. Quantitative real-time PCR was used to pro?le the change of transcriptional level of the two TIEG members in the mice tissues at six developmental stages. Taken together, the expression of TIEG1 and TIEG2 was speci?c in different organs yet varied with different developmental time points. Their dynamic changes were moderately consistent in most organs including the brain cortex, striatum, liver, kidney, and lung. However, their mRNA expression in both the heart and muscle was signi?cantly different at all developmental stages, which might propose a compensation of functions in the TIEG family. Nevertheless, our data indicate that both the TIEG genes are essential in regulating the normal organ development and functioning in murine model, as their expressions were ubiquitous and tissue speci?c at various developC 2010 Wiley-Liss, Inc. mental stages. Anat Rec, 293:858?864, 2010. V Key words: TIEG1; TIEG2; embryogenesis; mRNA expression Transforming growth factor-beta (TGF-b) and other growth factors constitute a large family of multifunctional proteins, which are known to regulate various bio- logical processes including cell growth, proliferation, differentiation, and apoptosis (Padgett et al., 1998; Chen and Meng, 2004). They are capable to induce various Lei Jianga and Yangchao Chen contributed equally to this work. Grant sponsors: Hong Kong Research Grants Council (GRF Grant); Grant numbers: CUHK462109, CUHK7422/03M, 467507; Grant sponsor: Special Grant of the Major State Basic Research Program of China; Grant number: 2006CB910100; Grant sponsor: Foundation of Guangzhou Science and Technology Bureau; Grant number: 2005Z1-E013; Grant sponsors: Chinese University of Hong Kong, Li Ka Shing Institute of Health Sciences. *Correspondence to: Ji-Cheng Li, Institute of Cell Biology, Zhejiang University, Hangzhou, 310058, Zhejiang Province, People?s Republic of China. E-mail: firstname.lastname@example.org. Phone: �-571-88208088; Fax: �-571-88208094. or H.-F. Kung, Stanley Ho Centre for Emerging Infectious Diseases, Rm511A, Basic Medical Sciences Building, The Chinese University of Hong Kong, Shatin, HKSAR. E-mail: email@example.com. Phone: �2-2603-7743; Fax: �2-2994-4988. Received 5 November 2008; Accepted 1 December 2009 DOI 10.1002/ar.21108 Published online 3 March 2010 in Wiley InterScience (www. interscience.wiley.com). C 2010 WILEY-LISS, INC. V TIEG1 AND TIEG2 EXPRESSION IN DEVELOPING MOUSE ORGANS cellular responses via particular receptor complex and Smad proteins, which depend on cell type and stimulation context (Rahimi and Leof, 2007). For example, they can induce growth arrest (i.e., apoptosis) in epithelial cells, which is a crucial step in suppressing tumors (Padgett et al., 1998; Sanchez et al., 1999; Cao et al., 2006). However, the TGF-b signaling pathway is also capable to promote carcinogenesis via induction of epithelial-mesenchymal transition (Rane et al., 2006; Caja et al., 2007). TGF-b-inducible early-response gene (TIEG) is a family of primary response genes induced by TGF-b and was originally identi?ed in human osteoblasts (Subramaniam et al., 1995). They are inducible by estrogen, an important anabolic hormone in the bone (Tau et al., 1998). TIEG gene encodes 480 amino acids and is regarded as one member of the Kru?ppel-like family of transcription factors (Fautsch et al., 1998; Chrisman and Tindall, 2003). TIEGs are involved in TFG-b signal transduction (Cook and Urrutia, 2000) and are playing signi?cant roles in regulating cell proliferation and apoptosis in various cell types (Tachibana et al., 1997; Ribeiro et al., 1999). Upon overexpression, TIEG1 enhanced TGF-b induction of Smad-binding element reporter activity (Johnsen et al., 2002a). Moreover, TIEG is thought to act as an inducer of gene transcription via upregulating the CD11d gene expression in myeloid cells (Noti et al., 2004). So far, three isoforms of TIEGs (TIEG1, TIEG2, and TIEG3) have been identi?ed. All of them contain three C2H2 zinc ?ngers near the C-terminus and one praline-rich N-terminal regulatory domain (Cook et al., 1999; Wang et al., 2004). All their mRNA expression can be upregulated in response to TGF-b1 treatment with similar induction time course (Cook et al., 1999; Hefferan et al., 2000b). Moreover, they have all been identi?ed in mouse (Yajima et al., 1997; Fautsch et al., 1998; Wang et al., 2004), whereas only two of them, TIEG1 and TIEG2, are identi?ed in human (Subramaniam et al., 1995; Cook et al., 1998). In general, TIEG2 shares 91% homology with TIEG1 within the zinc ?nger region (Cook et al., 1999), whereas TIEG3 shows 26 and 66% similarity to TIEG1 and TIEG2, respectively (Wang et al., 2004). Recently, signi?cant defect has been demonstrated in both osteoblasts and osteoclasts using a TIEG1 knockout mice model, which suggests a crucial role of TIEG1 in osteoblast function and osteoclast differentiation (Subramaniam et al., 2005). Using the same model in a later study, TIEG1 was shown to contribute signi?cantly at an age-dependent manner in the growth and maintenance of tendon microarchitecture and strength (Bensamoun et al., 2006b). In another study, TIEG null mice demonstrated severe and cardiac hypertrophy, which suggested a pivotal role of TIEG in normal cardiac development and functioning (Rajamannan et al., 2007). Moreover, TIEG1 overexpression was found to mimic TGF-b action in human osteoblast cells by increasing the alkaline phosphatase and decreasing osteocalcin secretion (Hefferan et al., 2000a). Similar mimicking effect was also observed in hepatocarcinoma (Ribeiro et al., 1999), pancreatic carcinoma (Tachibana et al., 1997), and mink lung epithelial cells (Chalaux et al., 1999), for TIEG1 overexpression would induce apoptosis and inhibit cell growth similar to that of TGF-b. The transcriptional level of TIEG1 was found 859 signi?cantly reduced in breast cancer, rendering it one of the most reliable markers to use with a sensitivity and speci?city of 96 and 93%, respectively (Reinholz et al., 2004). TIEG2 is a pancreas-enriched transcription factor, which can regulate exocrine cell growth and behaves as a tumor suppressor (Fernandez-Zapico et al., 2003). Recent studies have suggested TIEG2 as a potential endocrine regulator, while it might also play a pivotal role in postprandial glucose metabolism of skeletal muscle (Yamamoto et al., 2004). Besides, it can repress caveolin1 gene in adipose tissue in a cholesterol-dependent manner (Cao et al., 2005). Similar to TIEG1, the function of TIEG2 has also been investigated using knockout mice technique. However, no abnormalities were found in the knockout model; thus, it was believed that TIEG2 might not be a critical component in mice development (Song et al., 2005). In an earlier study, TIEG2 was shown to mimic the antiproliferative effects of TGF-b (Cook et al., 1998). Because of the strong sequence homology between TIEG1 and TIEG2, TIEG2 is also thought to involve in the regulation of apoptosis (Cook et al., 1998). Transient overexpression of TIEG2 has been reported to reduce the activity of Bcl-XL promoter and to decrease the BCL-XL protein level (Wang et al., 2007). It also induces Caspase3-dependent apoptosis in murine OLI-neu cells, which suggests its role as a downstream mediator of TGF-b that bridges the TGF-b signaling pathway with the apoptotic intracellular pathway (Wang et al., 2007). Although TIEGs are well recognized to regulate cellular proliferation and apoptosis, their expression pro?le has not been investigated during embryogenesis in various organs. In this study, we aim to investigate the transcriptional level of both TIEG1 and TIEG2 during development in various mice organs. Their exact roles in developmental process in different organs are discussed. MATERIALS AND METHODS Murine Embryos and Adult Tissues Embryos were carefully isolated from ICR mice, which were 12 (E12) and 16 (E16) days pregnant. Mice at various ages (i.e., 1 day, 8 days, 15 days, and adult mice) were sacri?ced by cervical dislocation. Individual tissues including brain cortex, cerebellum and stem (cere/stem), striatum, muscle, heart, liver, kidney, and lung were dissected. Tissues were collected and immediately immersed in liquid nitrogen. They were stored at 80 C until use. All experimental procedures were approved in prior by the Animal Experiment Ethics Committee of the Chinese University of Hong Kong. RNA Extraction and Reverse Transcription Total RNA was isolated using TRIZOL reagent (Invitrogen). Each RNA sample (2 lg) was reverse-transcribed using the ImProm-IITM Reverse transcription system (Promega) according to the manufacturer?s instructions. Quantitative Real-Time PCR The quantitation of mRNA was carried out using a real-time ?uorescence detection method. Quantitative real-time PCR (qRT-PCR) was performed using SYBRs 860 JIANG ET AL. Fig. 1. Relative amounts of TIEG1 mRNA expression in different organs during murine development. Gene expression value of E12 (BC � BS � CS) was set as 100%, and the values of the other samples were made relative to this value. E12, 12 day embryo; E16, 16 day embryo; P1, postnatal day 1; P8, postnatal day 8; P15, postnatal day 15; A, adult; BC, brain cortex; CS, cerebellum and stem; BS, brain striatum; L, liver; K, kidney; LU, lung; H, heart; M, muscle. GREEN PCR Master Mix (Applied Biosystems, Warrington, UK) and an ABI 7500 real-time PCR system (Applied Biosystems). DNA content was determined by measuring the real-time ?uorimetric intensity of SYBR green I incorporation after completion of the primer extension step in each cycle. A melting curve program was used to monitor the PCR product and to distinguish the samples from primer dimmers or other nonspeci?c products. Mock real-time PCR was also performed to evaluate genomic DNA contamination. A control housekeeping gene mouse glyceraldehyde-3-phosphate dehydrogenase (GADPH) was used as an internal control for normalizing variations due to differences in RNA quantity or ef?ciency of reverse transcription. The primers used in this study are listed as follows: TIEG1 forward primer: 50 -GCT CAA CTT CGG CGC TTC TC-30 , reverse primer: 50 -ACT TCC AGT CGC AGC TCA TG-30 ; TIEG2 forward primer: 50 -TCC CGA AGG AGG AAC TAT GT-30 , reverse primer: 50 -CCT GGG ATC TTC TTG GTT GT-30 ; GAPDH forward primer: 50 -AAC ATC AAA TGG GGT GAG GCC-30 , reverse primer: 50 -GTT GTC ATG GAT GAC CTT GGC-30 . The relative amount of mRNA expression of various samples was normalized to the level of GADPH. Standard expression curves for genes were also performed using a threefold dilution series of cDNAs derived from brain cortex of embryo of 12 days of age using RT-PCR. The expression level of both TIEG1 and TIEG2 in various tissues was divided by the corresponding expression level of GAPDH, thus to obtain the ?nal normalized value. In every group, the mRNA expression level of TIEG in the brain cortex of the Day 12 embryo (E12BC) was set as 100%, and each of their values found in other organs were compared and made relative to the E12BC value. Three independent experiments were carried out for each sample, and duplicate results were used to calculate the geometric mean. RESULTS Tissue-Speci?c TIEG1 and TIEG2 Expression During Murine Development As the brain cortex, brain striatum, and cere/stem were not well distinguished from each other at E12 stage, the whole brain tissues (marks as BC � BS � CS in the ?gures) were used for examining the TIEG level. In Figs. 1 and 2, they show the relative changes of TIEG1 and TIEG2 mRNA expression at different developmental stages in various organs, respectively. Based on our observation, TIEG1 and TIEG2 were expressed speci?cally in different organs yet varied with different developmental time points. Moreover, the expression level of TIEG1 in most of the organs at all of the developmental stages was signi?cantly higher than those of TIEG2. These might indicate that the TIEG1 is playing a relatively more crucial and essential role in regulating the development of murine organs than that of TIEG2. In Figs. 3 and 4, the data shown for the brain cortex, cere/stem, and brain striatum were their estimated values derived from the corresponding total TIEG1 or TIEG2 expression level in the brain, respectively. In TIEG1 AND TIEG2 EXPRESSION IN DEVELOPING MOUSE ORGANS Fig. 2. Relative amounts of TIEG2 mRNA expression in different organs during murine development. Fig. 3. Dynamic change of TIEG1 mRNA expression in various organs during murine development. 861 862 JIANG ET AL. Fig. 4. Dynamic change of TIEG2 mRNA expression in various organs during murine development. general, mRNA expression of TIEG1 and TIEG2 was low in the brain at all developmental stages. As seen in Fig. 3A, the brain cortex revealed a relatively higher expression of TIEG1 at P1 and P8. Similar pattern of expression was not followed by the cere/stem (Fig. 3B), yet could be observed in other brain compartment, such as the striatum with different extent (Fig. 3C). On the other hand, the TIEG2 gene appeared to be transiently induced in the brain cortex, cere/stem, and brain striatum at both the P1 and P8 stages (Fig. 4A?C). In the liver, the relative expression of TIEG1 and TIEG2 varied in similar pattern with their peak expressions noted at P1 (Fig. 3D) and P8 (Fig. 4D), respectively. Besides, the relative mRNA expression of TIEG1 kept increasing from E12 to P8 in the kidney, but signi?cantly lower expression was observed at P15 and adult (Fig. 3E). Similar pattern of increment was also observed for TIEG2 in the kidney (Fig. 4E). For TIEG1, a signi?cantly higher mRNA expression was observed in the lung with the peak expression recorded at P1 and then P8 (Fig. 3F), whereas highest level of TIEG2 was observed at P8 and then P1 (Fig. 3G). The expression level of TIEG2 is generally lower than that of TIEG1 at these two time points. In the heart, TIEG1 expressed exclusively higher in the mature/adult heart, whereas it remained at a very low level throughout development (Fig. 4F). For TIEG2, its mRNA level was very low in the heart at all stages including the mature heart (Fig. 4G). Finally, there was a markedly increase of TIEG1 in the muscle from E12 to adult, yet most signi?cant increase was noted between P8 and P15 (Fig. 3H). Reverse pattern was noted for TIEG2 in the muscle tissues with the highest of its expression noted at E12 and gradually decreased toward adolescence (Fig. 4H). Taken together, the dynamic changes of TIEG1 and TIEG2 expressions were relatively consistent in most organs including the brain cortex, striatum, liver, kidney, and lung, but not in the heart and muscle cells. DISCUSSION In this study, the mRNA expression of both TIEG1 and TIEG2 was pro?led during murine development. Their expression level in various organs was investigated using qRT-PCR at six developmental stages. According to our data, the mRNA expression of both TIEG1 and TIEG2 is tissue-speci?c manner at various developmental stages. Their expression patterns were quite similar in most organs including brain cortex, striatum, kidney, lung, and liver, but not in the heart and muscle. These might indicate a compensation of function in the TIEG family, particularly during the ?nal developmental stage of the cardiac and muscle cells. The ?ndings of this study provide the basic understanding on the expression pro?le of TIEG family during normal embryogenic and developmental stages. This is crucial if one needs to further investigate how and when to control these groups of proteins, which are closely related to TFG-b family, in combating different carcinogenic conditions, such as hepatocarcinoma and lung cancer. As seen in Fig. 1, low mRNA level of TIEG1 was found in different brain compartments and liver, whereas high level was expressed in the kidney during embryogenesis. It agrees with previous study in which TIEG1 was also observed in the brain, differentiating mesenchyme and kidney (Yajima et al., 1997). In fact, TIEG1 has been related to hippocampal network functioning via TGF-b TIEG1 AND TIEG2 EXPRESSION IN DEVELOPING MOUSE ORGANS signaling pathway (Lacmann et al., 2007). It was upregulated in somata of postsynaptic granule cells following both brain-derived neurotrophic factor- long-term potentiation and high-frequency stimulation-induced longterm potentiation (Wibrand et al., 2006). However, information of its role in brain development is still scarce. On the other hand, TIEG1 is known to be regulated by connective TGF in human mesangial cells, thus enhanced the TGF-b signaling pathway (Wahab et al., 2005). In hepatal tissues, TIEG1 has been reported to induce apoptosis in hepatoma Hep3B cells (Ribeiro et al., 1999). In addition, it was known to repress glutathione transferase P gene expression in rat liver and was found to be useful in suppressing early stage of chemical hepatocarcinogenesis (Tanabe et al., 2002). However, the exact role of TIEG1 in normal renal and hepatal development is yet to be elucidated. As mentioned earlier, TIEG1 is known to mimic TGF-b action in mink lung epithelial cells (Chalaux et al., 1999). Overexpression of TIEG1 may decrease endogenous Bcl-2 levels and elicit programed cell death. In this study, the high expression of TIEG1 at the P1 and P8 stages may indicate a stimulated apoptotic cell death in this organ at this period of development. It could be a physiological variation that used to mediate various cell growth and proliferation at particular interval of murine development. Such upregulation could also be a response to the stimulation of other factors, such as bone-morphogenetic protein-2 (Hefferan et al., 2000b) or estrogen (Tau et al., 1998). The high expression level of TIEG in the lung may indicate high apoptotic rate, which could explain the high regenerative activity in the lungs of younger mice. In previous studies, TIEG1 was reported to express in normal human myocardium (Subramaniam et al., 1995, 1998). The signaling pathway of TIEG1 was thought to implicate cardiomyocyte growth and ?brosis (Li et al., 1998), while absence of the gene resulted in cardiac hypertrophy (Rajamannan et al., 2007). Therefore, normal expression level of TIEG is undoubtedly pivotal for normal heart development, which might explain the high expression level of TIEG in the mature mice heart. High TIEG expression has been reported in skeletal tissues and human osteoblasts (Subramaniam et al., 1995). TIEG is believed to be a key regulatory factor in the TGFb action in the tissues. In addition, it was suggested to associate with Src homology-3 in the signal transduction processes (Subramaniam et al., 1995). In an earlier TIEG1 null mice model, the animals were physically weaker than those of the wild types (Bensamoun et al., 2006a). Previous study has also revealed a drastic drop of bone content, density, and size in the animal model. Electron microscopy also demonstrated a signi?cant decrease in osteocyte number in the TIEG1 knock out mice model, which suggests a crucial role of TIEG1 in osteoblast differentiation. It is generally believed that TIEG1 is crucial for healthy bone development (Bensamoun et al., 2006a). Therefore, the exact functions of TIEGs in the muscle tissues warrant further investigations. In this study, the expression of both TIEG isoforms was generally low and steady throughout the developmental stages. In the oligodendroglial cell line, OLI-neu, overexpression of TIEG has been found to downregulate the protein expression of Bcl-2 family and to reduce the antiapoptotic mediator, Bcl-XL, at both transcription and translational levels (Bender et al., 2004). The induced 863 repression is nicely parallel with the TGF-b-induced apoptosis, which strongly indicates that the TIEG is a downstream protein in the TGF-b-induced cell death. On the other hand, Bcl-2 family members are known to play a critical role in embryonic development. Recently, TIEG2 has been reported to downregulate one of the Bcl-2 family members, Bcl-XL, thus led to caspase-3-dependent apoptosis (Wang et al., 2007). Therefore, TIEG2 might act directly in regulating cell proliferation and apoptosis via enhancing the TGF-b signaling pathway, yet they might also function through mediating the Bcl-2 family proteins. In the liver, TIEG2 was involved in complete gene regulation by functioning as an activator, that increased monoamine oxidases B gene expression at promoter, mRNA, protein, and catalytic activity levels in both the SH-SY5Y and HepG2 cells (Ou et al., 2004). TIEG2 is also known to express ubiquitously in human tissues, with enrichment in pancreas and muscle (Cook et al., 1998). However, its functional role during embryogenesis and normal development has not been reported in organs. With a 91% homology with TIEG1 within the zinc ?nger region and 44% homology within the N terminus, it might be reasonable to postulate that TIEG2 shares similar function as TIEG1, but differs in their scope of actions. Nevertheless, TIEG is believed to act as repressor of Smad7 (Johnsen et al., 2002b) and an enhancer for SBE promoter activity (Bender et al., 2004), which exempli?es the complex functions of the TIEG proteins. In this study, qRT-PCR is used to investigate the TIEG expression in various organs. In general, this method is a highly sensitive and speci?c technique, but sometimes the results could be misleading at speci?c conditions. For example, a high-level expression in a small subset of cells or cell types in a particular tissue could be underestimated, or the transcriptional changes might not be evident in the tissues yet overampli?cation could be unappreciated by the qRT-PCR. Therefore, a whole-mount in situ hybridization approach may be more appropriate in this case (i.e., at early stages of embryogenesis). Taken together, the expression of TIEG1 and TIEG2 is speci?c in different organs yet varied with different developmental time points. Their dynamic changes of expression during murine development are consistent in most organs except in the heart and muscle tissues, which indicate a compensation of functions in the TIEG family. Further investigations would be necessary to clarify this issue and their roles in murine organogenesis. LITERATURE CITED Bender H, Wang Z, Schuster N, Krieglstein K. 2004. TIEG1 facilitates transforming growth factor-beta-mediated apoptosis in the oligodendroglial cell line OLI-neu. J Neurosci Res 75:344?352. Bensamoun SF, Hawse JR, Subramaniam M, Ilharreborde B, Bassillais A, Benhamou CL, Fraser DG, Oursler MJ, Amadio PC, An KN, Spelsberg TC. 2006a. TGFbeta inducible early gene-1 knockout mice display defects in bone strength and microarchitecture. Bone 39:1244?1251. Bensamoun SF, Tsubone T, Subramaniam M, Hawse JR, Boumediene E, Spelsberg TC, An KN, Amadio PC. 2006b. Age-dependent changes in the mechanical properties of tail tendons in TGF-beta inducible early gene-1 knockout mice. J Appl Physiol 101:1419?1424. Caja L, Ortiz C, Bertran E, Murillo MM, Miro-Obradors MJ, Palacios E, Fabregat I. 2007. Differential intracellular signalling induced by TGF-beta in rat adult hepatocytes and hepatoma cells: implications in liver carcinogenesis. Cell Signal 19:683?694. 864 JIANG ET AL. Cao S, Fernandez-Zapico ME, Jin D, Puri V, Cook TA, Lerman LO, Zhu XY, Urrutia R, Shah V. 2005. KLF11-mediated repression antagonizes Sp1/sterol-responsive element-binding proteininduced transcriptional activation of caveolin-1 in response to cholesterol signaling. J Biol Chem 280:1901?1910. Cao Y, Deng C, Townsend CM, Jr., Ko TC. 2006. TGF-beta inhibits Akt-induced transformation in intestinal epithelial cells. Surgery 140:322?329. Chalaux E, Lopez-Rovira T, Rosa JL, Pons G, Boxer LM, Bartrons R, Ventura F. 1999. A zinc-?nger transcription factor induced by TGF-beta promotes apoptotic cell death in epithelial Mv1Lu cells. FEBS Lett 457:478?482. Chen YG, Meng AM. 2004. Negative regulation of TGF-beta signaling in development. Cell Res 14:441?449. Chrisman HR, Tindall DJ. 2003. Identi?cation and characterization of a consensus DNA binding element for the zinc ?nger transcription factor TIEG/EGRalpha. DNA Cell Biol 22:187?199. Cook T, Gebelein B, Belal M, Mesa K, Urrutia R. 1999. Three conserved transcriptional repressor domains are a de?ning feature of the TIEG subfamily of Sp1-like zinc ?nger proteins. J Biol Chem 274:29500?29504. Cook T, Gebelein B, Mesa K, Mladek A, Urrutia R. 1998. Molecular cloning and characterization of TIEG2 reveals a new subfamily of transforming growth factor-beta-inducible Sp1-like zinc ?ngerencoding genes involved in the regulation of cell growth. J Biol Chem 273:25929?25936. Cook T, Urrutia R. 2000. TIEG proteins join the Smads as TGFbeta-regulated transcription factors that control pancreatic cell growth. Am J Physiol Gastrointest Liver Physiol 278:G513? G521. Fautsch MP, Vrabel A, Rickard D, Subramaniam M, Spelsberg TC, Wieben ED. 1998. Characterization of the mouse TGFbeta-inducible early gene (TIEG): conservation of exon and transcriptional regulatory sequences with evidence of additional transcripts. Mamm Genome 9:838?842. Fernandez-Zapico ME, Mladek A, Ellenrieder V, Folch-Puy E, Miller L, Urrutia R. 2003. An mSin3A interaction domain links the transcriptional activity of KLF11 with its role in growth regulation. EMBO J 22:4748?4758. Hefferan TE, Reinholz GG, Rickard DJ, Johnsen SA, Waters KM, Subramaniam M, Spelsberg TC. 2000a. Overexpression of a nuclear protein, TIEG, mimics transforming growth factor-beta action in human osteoblast cells. J Biol Chem 275:20255?20259. Hefferan TE, Subramaniam M, Khosla S, Riggs BL, Spelsberg TC. 2000b. Cytokine-speci?c induction of the TGF-beta inducible early gene (TIEG): regulation by speci?c members of the TGF-beta family. J Cell Biochem 78:380?390. Johnsen SA, Subramaniam M, Janknecht R, Spelsberg TC. 2002a. TGFbeta inducible early gene enhances TGFbeta/Smad-dependent transcriptional responses. Oncogene 21:5783?5790. Johnsen SA, Subramaniam M, Katagiri T, Janknecht R, Spelsberg TC. 2002b. Transcriptional regulation of Smad2 is required for enhancement of TGFbeta/Smad signaling by TGFbeta inducible early gene. J Cell Biochem 87:233?241. Lacmann A, Hess D, Gohla G, Roussa E, Krieglstein K. 2007. Activity-dependent release of transforming growth factor-beta in a neuronal network in vitro. Neuroscience 150:647?657. Li G, Li RK, Mickle DA, Weisel RD, Merante F, Ball WT, Christakis GT, Cusimano RJ, Williams WG. 1998. Elevated insulin-like growth factor-I and transforming growth factor-beta 1 and their receptors in patients with idiopathic hypertrophic obstructive cardiomyopathy. A possible mechanism. Circulation 98:II144?II149; discussion II149?II150. Noti JD, Johnson AK, Dillon JD. 2004. The zinc ?nger transcription factor transforming growth factor beta-inducible early gene-1 confers myeloid-speci?c activation of the leukocyte integrin CD11d promoter. J Biol Chem 279:26948?26958. Ou XM, Chen K, Shih JC. ( 2004). Dual functions of transcription factors, transforming growth factor-beta-inducible early gene (TIEG)2 and Sp3, are mediated by CACCC element and Sp1 sites of human monoamine oxidase (MAO) B gene. J Biol Chem 279: 21021?21028. Padgett RW, Das P, Krishna S. 1998. TGF-beta signaling, Smads, and tumor suppressors. Bioessays 20:382?390. Rahimi RA, Leof EB. 2007. TGF-beta signaling: a tale of two responses. J Cell Biochem 102:593?608. Rajamannan NM, Subramaniam M, Abraham TP, Vasile VC, Ackerman MJ, Monroe DG, Chew TL, Spelsberg TC. 2007. TGFbeta inducible early gene-1 (TIEG1) and cardiac hypertrophy: discovery and characterization of a novel signaling pathway. J Cell Biochem 100:315?325. Rane SG, Lee JH, Lin HM. 2006. Transforming growth factor-beta pathway: role in pancreas development and pancreatic disease. Cytokine Growth Factor Rev 17:107?119. Reinholz MM, An MW, Johnsen SA, Subramaniam M, Suman VJ, Ingle JN, Roche PC, Spelsberg TC. 2004. Differential gene expression of TGF beta inducible early gene (TIEG), Smad7, Smad2 and Bard1 in normal and malignant breast tissue. Breast Cancer Res Treat 86:75?88. Ribeiro A, Bronk SF, Roberts PJ, Urrutia R, Gores GJ. 1999. The transforming growth factor beta(1)-inducible transcription factor TIEG1, mediates apoptosis through oxidative stress. Hepatology 30:1490?1497. Sanchez A, Alvarez AM, Lopez Pedrosa JM, Roncero C, Benito M, Fabregat I. 1999. Apoptotic response to TGF-beta in fetal hepatocytes depends upon their state of differentiation. Exp Cell Res 252:281?291. Song CZ, Gavriilidis G, Asano H, Stamatoyannopoulos G. 2005. Functional study of transcription factor KLF11 by targeted gene inactivation. Blood Cells Mol Dis 34:53?59. Subramaniam M, Gorny G, Johnsen SA, Monroe DG, Evans GL, Fraser DG, Rickard DJ, Rasmussen K, van Deursen JM, Turner RT, Oursler MJ, Spelsberg TC. 2005. TIEG1 null mouse-derived osteoblasts are defective in mineralization and in support of osteoclast differentiation in vitro. Mol Cell Biol 25:1191?1199. Subramaniam M, Harris SA, Oursler MJ, Rasmussen K, Riggs BL, Spelsberg TC. 1995. Identi?cation of a novel TGF-beta-regulated gene encoding a putative zinc ?nger protein in human osteoblasts. Nucleic Acids Res 23:4907?4912. Subramaniam M, Hefferan TE, Tau K, Peus D, Pittelkow M, Jalal S, Riggs BL, Roche P, Spelsberg TC. 1998. Tissue, cell type, and breast cancer stage-speci?c expression of a TGF-beta inducible early transcription factor gene. J Cell Biochem 68:226?236. Tachibana I, Imoto M, Adjei PN, Gores GJ, Subramaniam M, Spelsberg TC, Urrutia R. 1997. Overexpression of the TGFbeta-regulated zinc ?nger encoding gene, TIEG, induces apoptosis in pancreatic epithelial cells. J Clin Invest 99:2365?2374. Tanabe A, Kurita M, Oshima K, Osada S, Nishihara T, Imagawa M. 2002. Functional analysis of zinc ?nger proteins that bind to the silencer element in the glutathione transferase P gene. Biol Pharm Bull 25:970?974. Tau KR, Hefferan TE, Waters KM, Robinson JA, Subramaniam M, Riggs BL, Spelsberg TC. 1998. Estrogen regulation of a transforming growth factor-beta inducible early gene that inhibits deoxyribonucleic acid synthesis in human osteoblasts. Endocrinology 139:1346?1353. Wahab NA, Weston BS, Mason RM. 2005. Modulation of the TGFbeta/Smad signaling pathway in mesangial cells by CTGF/ CCN2. Exp Cell Res 307:305?314. Wang Z, Peters B, Klussmann S, Bender H, Herb A, Krieglstein K. 2004. Gene structure and evolution of Tieg3, a new member of the Tieg family of proteins. Gene 325:25?34. Wang Z, Spittau B, Behrendt M, Peters B, Krieglstein K. 2007. Human TIEG2/KLF11 induces oligodendroglial cell death by downregulation of Bcl-X(L) expression. J Neural Transm 114:867?875. Wibrand K, Messaoudi E, Havik B, et al. 2006. Identi?cation of genes co-upregulated with Arc during BDNF-induced long-term potentiation in adult rat dentate gyrus in vivo. Eur J Neurosci 23:1501?1511. Yajima S, Lammers CH, Lee SH, Hara Y, Mizuno K, Mouradian MM. 1997. Cloning and characterization of murine glial cellderived neurotrophic factor inducible transcription factor (MGIF). J Neurosci 17:8657?8666. Yamamoto J, Ikeda Y, Iguchi H, Fujino T, Tanaka T, Asaba H, Iwasaki S, Ioka RX, Kaneko IW, Magoori K, Takahashi S, Mori T, Sakaue H, Kodama T, Yanagisawa M, Yamamoto TT, Ito S, Sakai J. 2004. A Kruppel-like factor KLF15 contributes fasting-induced transcriptional activation of mitochondrial acetyl-CoA synthetase gene AceCS2. J Biol Chem 279:16954?16962.