TGF-beta Inducible Early Gene Research Articles

Bone marrow stroma cells regulate TIEG1 expression in acute lymphoblastic leukemia cells: Role of TGFβ/BMP‐6 and TIEG1 in chemotherapy escape

The bone marrow microenvironment regulates early B lymphopoiesis and protects leukemia cells against chemotherapy treatment, thus the microenvironment may serve as a sanctuary site for these cells. Yet, few factors that contribute to this process are known. We have explored the role of transforming growth factor beta (TGFbeta) and bone morphogenetic protein-6 (BMP-6) and one target gene, TGFbeta inducible early gene 1 (TIEG1), in the communication between stroma cells and acute lymphoblastic leukemia (ALL) cell lines and their escape from chemotherapy. Here, we have demonstrated TIEG1 expression in both normal B progenitor cells and ALL cells, which increased rapidly upon TGFbeta and BMP-6 treatment. Stimulation with TGFbeta or BMP-6, as well as overexpression of TIEG1 inhibited proliferation. Furthermore, interaction with stroma cells induced TIEG1 expression in ALL cells, inhibited their proliferation and protected the cells against chemotherapeutic treatment. Similarly, treatment with TGFbeta or BMP-6, as well as overexpression of TIEG1, protected ALL cells against chemotherapy-induced cell death. These data suggest that TGFbeta and BMP-6 in the bone marrow microenvironment allow leukemia cells to escape therapy. Further, the data indicate that TIEG1 might be involved in mediating this effect from the microenvironment onto the leukemia cells.

TGFβ inducible early gene‐1 (TIEG1) and cardiac hypertrophy: Discovery and characterization of a novel signaling pathway

Cellular mechanisms causing cardiac hypertrophy are currently under intense investigation. We report a novel finding in the TGFbeta inducible early gene (TIEG) null mouse implicating TIEG1 in cardiac hypertrophy. The TIEG(-/-) knock-out mouse was studied. Male mice age 4-16 months were characterized (N = 86 total) using echocardiography, transcript profiling by gene microarray, and immunohistochemistry localized upregulated genes for determination of cellular mechanism. The female mice (N = 40) did not develop hypertrophy or fibrosis. The TIEG(-/-) knock-out mouse developed features of cardiac hypertrophy including asymmetric septal hypertrophy, an increase in ventricular size at age 16 months, an increase (214%) in mouse heart/weight body weight ratio TIEG(-/-), and an increase in wall thickness in TIEG(-/-) mice of (1.85 +/- 0.21 mm), compared to the control (1.13 +/- 0.15 mm, P < 0.04). Masson Trichrome staining demonstrated evidence of myocyte disarray and myofibroblast fibrosis. Microarray analysis of the left ventricles demonstrated that TIEG(-/-) heart tissues expressed a 13.81-fold increase in pituitary tumor-transforming gene-1 (Pttg1). An increase in Pttg1 and histone H3 protein levels were confirmed in the TIEG(-/-) mice hearts tissues. We present evidence implicating TIEG and possibly its target gene, Pttg1, in the development of cardiac hypertrophy in the TIEG null mouse.

Effect of TGF‐β inducible early gene deficiency on flexor tendon healing

The role of transforming growth factor beta (TGF-beta) in tendon healing is still not clearly established. TGF-beta affects gene expression primarily through the activation of the Smad signaling pathway. The first step in the Smad pathway is the expression of TGF-beta inducible early gene (TIEG). Recently, a TIEG knockout mouse has been developed. The purpose of this study was to examine the healing potential of flexor tendons in mice lacking the TIEG gene, and to further examine what role the TIEG pathway plays in flexor tendon repair. Twenty-two mice, consisting of 11 normal wild-type mice and 11 TIEG knockout mice, were euthanized at 8 to 12 weeks of age. The second through fifth FDL tendons of both hind feet were transected and repaired in zone 2. The repaired tendons were removed from the mice and placed into tissue culture. Tendons were then examined at days 3, 7, 14, 21, and 42 after surgery. Hematoxylin and eosin (HE) staining and immunohistochemical staining for TGF-beta, collagen type I, and collagen type III were performed. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed to examine expression of TGF-beta1, beta2, beta3, and collagen type I and III. At 42 days after surgery, HE staining showed coaptation of lacerated tendon ends in both groups. Both groups showed healing of the lacerated tendon, but the chronologic expression pattern of TGF-beta was different between the knockout and normal tendons. TIEG deficient tendons had delayed expression of TGF-beta when compared with control tendons. The collagen mRNA expression pattern was similar with both groups, but the expression level was different, with TIEG knockout tendons having a lower expression of collagen type I mRNA (p < 0.001). TGF-beta is thought to play a major role in tendon healing. Healing of tendons in the TIEG knockout mouse suggests the possibility of tendon healing in the absence of the Smad pathway. The knockout mouse model described in the present study provides a novel means for further understanding of the tendon healing process through isolated deletion of specific growth factors.

325. Identification of Mechanistically Distinct Repression Domains at Endogenous Genes Using Engineered Zinc Finger Proteins

Engineered zinc finger protein transcription factors (ZFP TFs) are capable of controlling the expression of targeted endogenous genes, and thus provide potential therapeutics for the treatment of disease. The functionality of ZFP TFs results from the modularity of the DNA binding and effector domains allowing such ZFP-fusion proteins to address a given domain to a specific chromosomal gene in vivo. This technology thus allows novel effector domains to be assayed for function directly at an endogenous gene. Since this system requires no insertion of binding sites or other foreign DNA into the region of interest, the efficacy of a given ZFP-domain fusion can be determined in the context of truly native chromatin. This is of particular significance to transcriptional regulation since promoter specific modification of histones plays a dynamic role in the process of gene control. We show here that a ZFP targeted to an endogenous reporter gene can be used to successfully identify new effector domains capable of repressing gene expression in vivo. Furthermore we show that the requirements for gene repression at an endogenous gene are not always recapitulated by more standard plasmid-based reporter assays. Finally we use this system to identify different effector domains that employ alternative repression pathways, and show that a domain from the TGF-beta inducible early gene is capable of driving gene repression in an HDAC and DNA methylation independent manner. The ability to screen for, and employ alternative effector domains with defined sensitivity toward known drugs opens the potential for the combinatorial use of such compounds with ZFP TF therapeutic interventions to both accentuate, or limit their function.

15-hydroxy-eicosatetraenoic acid arrests growth of colorectal cancer cells via a peroxisome proliferator-activated receptor gamma-dependent pathway.

Peroxisome proliferator-activated receptor gamma (PPARgamma) inhibits cell growth via promoting apoptosis. Human colorectal cancer tissues had abundant PPARgamma but the incidence of apoptosis was very low, suggesting a defect in the PPARgamma pathway. Here, we found that 15-hydroxy-eicosatetraenoic acid (15S-HETE), an endogenous ligand for PPARgamma, was significantly decreased in the serum of patients with colorectal cancer. Treatment of colon cancer cells with 15S-HETE inhibited cell proliferation and induced apoptosis, which was preceded by an increase in TGF-beta-inducible early gene (TIEG) and a decrease in Bcl-2. The action of 15S-HETE could be blocked when PPARgamma was suppressed. Overexpression of Bcl-2 prevented the apoptosis. The levels of TIEG and 15-lipoxygenase (15-LOX), the enzyme responsible for 15S-HETE production, was decreased in colorectal cancer. Therefore, colorectal cancer is associated with decreased 15S-HETE. Treatment of colon cancer cells with 15S-HETE inhibits cell proliferation and induces apoptosis in a PPARgamma-dependent pathway involving augmentation of TIEG and reduction of Bcl-2 expression.

Identification and characterization of a consensus DNA binding element for the zinc finger transcription factor TIEG/EGRalpha.

TGFbeta-Inducible Early Gene (TIEG) and the alternatively-transcribed Early Growth Response Gene alpha (EGRalpha) share a Cys(2)His(2) three-zinc finger region with high homology to Sp1 within its zinc finger region. Three-zinc finger transcription factors bind to GC-rich sequences, with small variations in consensus sequence between subfamilies. In this work, a consensus sequence was identified for TIEG/EGRalpha by expressing and purifying the zinc finger region of the protein, and using this to select a binding site from a random oligonucleotide library by iterative cycles of nitrocellulose filter binding and PCR. A fusion of the TIEG/EGRalpha with the VP16 activation domain supported transcription from this site when cloned in front of a heterologous promoter. Mutational analysis of the binding site identified a GT-rich core (5'-GGTGTG-3') that was necessary for binding, with mutations outside of this region causing only a small to moderate decrease in binding.

TGFbeta inducible early gene enhances TGFbeta/Smad-dependent transcriptional responses.

TGFbeta inducible early gene (TIEG) encodes a three zinc-finger Krüppel-like transcription factor whose overexpression has been shown to mimic the effects of TGFbeta in human osteosarcoma and pancreatic carcinoma cells. In order to investigate a potential role of TIEG in the TGFbeta signal transduction pathway, we studied its impact on a Smad binding element (SBE) reporter which is known to be regulated by TGFbeta through the R-Smad proteins. We demonstrate that TIEG overexpression enhances TGFbeta induction of SBE reporter activity. TIEG overexpression also enhances induction of the endogenous TGFbeta regulated genes p21 and PAI-1. The ability of TIEG to enhance TGFbeta actions is Smad dependent since TIEG has no effect on SBE transcription in the absence of Smad4 expression or when an inhibitory Smad protein, Smad7, is overexpressed. Furthermore, TIEG overexpression enhances TGFbeta induced Smad2 phosphorylation. Lastly, TIEG appears to function by binding to and thereby repressing a specific element in the proximal promoter of the inhibitory Smad7 gene. In conclusion, these results describe a novel mechanism for the potentiation of TGFbeta/Smad signaling via repression of the inhibitory Smad7 gene by TIEG.

Transcriptional regulation of Smad2 is required for enhancement of TGFbeta/Smad signaling by TGFbeta inducible early gene.

TGFbeta inducible early gene (TIEG) is a novel Krüppel-like transcriptional repressor that was recently shown to increase the activity of the TGFbeta/Smad signal transduction pathway by relieving negative feedback through repression of the inhibitory Smad7. Interestingly, while Smad7 is required for maximal enhancement of TGFbeta/Smad signaling, we observe that TIEG is still capable of increasing Smad pathway activity in the absence of Smad7. Furthermore, while Smad7 is known to block both TGFbeta and bone morphogenetic protein (BMP) signaling, we observe that TIEG specifically enhances only the TGFbeta pathway. Similarly, while both TIEG and the related Krüppel-like factor, FKLF2, repress Smad7 transcription, only TIEG is capable of enhancing Smad signaling. In order to identify additional regulatory targets of TIEG important for this enhancement of the Smad pathway activity, we performed microarray analysis and identified Smad2 as a TIEG target gene. We now show evidence that TIEG increases transcription of the Smad2 gene but not the Smad3 or Smad4 genes. Furthermore, while the TGFbeta/Smad pathway remains intact in Smad2 null cells, TIEG enhancement of Smad signaling is dramatically reduced. Thus we propose a new model whereby TIEG enhances Smad signaling by a dual mechanism involving both the repression of the inhibitory Smad7 as well as the activation of Smad2.

TGFβ-mediated signaling and transcriptional regulation in pancreatic development and cancer

Transforming growth factor-beta (TGFbeta) plays a critical role in pancreatic development and cell proliferation. Binding of TGFbeta to its membrane receptor kinases activates the Smad signaling proteins, allowing them to translocate to the nucleus and participate in the transcriptional control of TGFbeta target genes. In addition, there is an increasing number of cellular mechanisms affecting the final response of a cell to TGFbeta. This includes crosstalk with other signaling pathways and the induction of TGFbeta early response genes, such as the TGFbeta-inducible early response gene (TIEG) family of transcription factors. Like the Smads, TIEGs behave as downstream effector proteins in TGFbeta-mediated pancreatic growth control. The discovery of the Smads and TIEGs has provided new insights into TGFbeta-regulated functions. Their significance in pancreatic development and cancer is discussed in this review.

Transforming growth factor-beta1-induced Smad signaling, cell-cycle arrest and apoptosis in hepatoma cells

Transforming growth factor-beta1 (TGFbeta) is involved in the regulation of liver cell proliferation and apoptosis, and escape of hepatoma cells from the growth restraining signals of TGFbeta has been suggested to contribute to tumor development. TGFbeta modulates gene transcription by receptor-mediated activation of Smad proteins which act as transcription factors. TGFbeta-mediated primary signaling responses as well as effects on the cell cycle and apoptosis were investigated in the human hepatoblastoma line HepG2, the rat hepatoma line FTO-2B and the mouse hepatoma line 55.1c. Activation of a Smad (Sma and Mad homolog) response-element-driven luciferase reporter by TGFbeta was very similar in all three cell lines, indicating functionality of the primary TGFbeta signaling pathway. Moreover, TGFbeta-inducible early gene was transiently activated by TGFbeta in all cell lines as shown by RT-PCR. HepG2 cells, however, were completely resistant to TGFbeta-induced growth arrest and apoptosis and 55.1c cells were only slightly susceptible to TGFbeta-induced apoptosis. By contrast, treatment of FTO-2B cells with TGFbeta led to a partial G0/G1 arrest and a strong induction of apoptosis. TGFbeta-induced apoptosis of FTO-2B cells was inhibited by dexamethasone, insulin, phenobarbital and dieldrin. Of these agents, only insulin led to a significant reduction of TGFbeta-stimulated Smad-reporter activity, suggesting that the other compounds interfere with TGFbeta-induced apoptosis downstream of Smad-mediated primary transcriptional responses at a level that may be constitutively altered in apoptosis-resistant hepatoma cell lines.

Overexpression of a Nuclear Protein, TIEG, Mimics Transforming Growth Factor-β Action in Human Osteoblast Cells

Although transforming growth factor-beta (TGF-beta) is a growth factor with many known regulatory activities in many different cell types, its intracellular signaling pathway is still not fully understood. A TGF-beta-inducible early gene (TIEG) was discovered and shown by this laboratory to be a 3-zinc finger transcription factor family member; its expression is rapidly induced in cells treated with TGF-beta. To ascertain whether TIEG plays a major role in the TGF-beta pathway, human osteosarcoma MG-63 cells were stably transfected either with an expression vector containing a TIEG cDNA or with the vector alone. Clones that contain only the vector express normal levels of TIEG mRNA and protein and display the same patterns of gene expression and levels of cell proliferation as the nontransfected, non-TGF-beta-treated parental cells. However, transfected cells that overexpress TIEG mRNA and protein (TIEG-6 and TIEG-7) display changes that mimic those of MG-63 cells treated with TGF-beta, i.e. increased alkaline phosphatase activity, decreased levels of osteocalcin mRNA and protein, and decreased cell proliferation. The degree of these changes correlated with the level of TIEG expressed in the cell lines. TGF-beta treatment of the overexpressed cells showed no added effects. These findings and other published reports support a primary role of TIEG as a transcription factor in the TGF-beta signaling pathway.

Direct action of naturally occurring estrogen metabolites on human osteoblastic cells.

This article describes experiments that were performed to examine the direct action of estrogen metabolites on cultured human osteoblast cells. The human fetal osteoblastic cell line, hFOB/ER9, which expresses high levels of the estrogen receptor (ER) alpha, was used to examine the direct effects of 16alpha-hydroxyestrone (16alpha-OHE1) and 2-hydroxyestrone (2-OHE1) on osteoblast differentiation. The 16alpha-OHE1 caused a decrease in osteocalcin (OC) secretion to a maximum of 40% of control values (vehicle-treated cells) at 10(-7) M. Alkaline phosphatase (AP) activity was significantly induced at 10(-7) M 16alpha-OHE1 with greater than 500% of control at 10(-6) M 16alpha-OHE1. Finally, AP steady-state messenger RNA (mRNA) levels were increased within 24 h of 16alpha-OHE1 treatment. In contrast to 16alpha-OHE1, 2-OHE1 had no effects on the secretion of OC, AP activity, or AP gene expression. The 2-OHE1 also did not display any antiestrogen activity because treatment in combination with 17beta-estradiol (E2) and 16alpha-OHE1 had no significant effect on the reduction in OC secretion or induction of AP activity. Similar to E2, 16alpha-OHE1 stimulated the expression of an early response gene, a TGF-beta inducible early gene, designated TIEG, as early as 60 minutes after treatment, whereas treatment with 2-OHE1 displayed no effect. Support that the 16alpha-OHE1 regulation of these osteoblasts (OB) markers was mediated through the ER is shown by the fact that the estrogen antagonist ICI 182,780 abrogated these effects. These data suggest that is a potent estrogen agonist on human osteoblastic hOB/ER9 cells. In contrast, 2-OHE1 displayed no estrogenic or antiestrogenic activity in this human osteoblast cell model.

Cytokine-specific induction of the TGF-beta inducible early gene (TIEG): regulation by specific members of the TGF-beta family.

Select members of the TGF-beta family of cytokines play key regulatory roles in skeletal development, structure, and turnover. This laboratory has previously reported that TGF-beta treatment of immortalized normal human fetal osteoblast (hFOB) cells results in the rapid induction of the mRNA levels of a TGF-beta inducible early gene (TIEG) followed by changes in cell proliferation and bone matrix protein production. Previous studies have also shown that nonmembers of the TGF-beta superfamily showed little or no induction of TIEG mRNA. This article further addresses the cytokine specificity of this TIEG induction by examining whether activin and select bone morphogenetic proteins, (BMP-2, BMP-4, and BMP-6), which are representative of different subfamilies of this superfamily, also induce the expression of TIEG in hFOB cells. However, TGF-beta remained the most potent of these cytokines, inducing TIEG mRNA steady-state levels at 0.1 ng/ml, with a maximum induction of 24-fold at 2.0 ng/ml. The BMP-2 (16-fold), BMP-4 (4-fold), and activin (1-3-fold) also induced TIEG mRNA levels, but at reduced degrees compared to TGF-beta (24-fold), and only at much higher cytokine concentrations, e.g., 50-100 ng/ml, compared to 2 ng/ml for TGF-beta. BMP-6 showed no effect on TIEG mRNA levels. The TIEG protein levels generally correlated with the mRNA steady-state levels. As with TGF-beta, BMP-2 treatment of hFOB cells was shown by confocal microscopy to induce a rapid translocation of the TIEG protein to the nucleus. In summary, the relative potencies of these TGF-beta family members to induce TIEG expression generally follows the general osteoinductive capacity of these cytokines, with TGF-beta >>> BMP-2 > BMP-4 > activin >> BMP-6.

Tissue, cell type, and breast cancer stage-specific expression of a TGF-β inducible early transcription factor gene

This laboratory has previously identified a novel TGF-beta inducible early gene (TIEG) in human osteoblasts [Subramaniam et al. (1995): Nucleic Acids Res 23:4907-4912]. Using TIEG specific polyclonal antibody and immunoprecipitation methods in normal human fetal osteoblast cells (hFOB cells), we have now demonstrated that TIEG encodes a 72-kDa protein whose levels are transiently increased at as early as 2 h of TGF-beta treatment. Polarized confocal microscopic analysis of hFOB cells shows a nuclear localized TIEG protein in untreated cells under the conditions described under Methods. Interestingly, the levels of TIEG protein in the nuclei increase when the cells are treated with TGF-beta 1 for 2 h. In contrast, similar analyses of untreated human keratinocytes show a cytoplasmic localized TIEG protein that appears to be translocated to the nucleus after H2O2 treatment. Additional immunohistochemical studies have demonstrated that TIEG protein is expressed in epithelial cells of the placenta, breast, and pancreas, as well as in osteoblast cells of bone and selected other cells of the bone marrow and cerebellum with some cells showing a cytoplasmic localization and others a nuclear localization. All cells of the kidney display negative staining for this protein. Interestingly, a stage specific expression of TIEG protein is found in a dozen breast cancer biopsies, using immunohistochemistry. The cells in normal breast epithelium displays a high expression of TIEG protein, those in the in situ carcinoma display less than one-half of the levels, and those in the invasive carcinoma show a complete absence of the TIEG protein. TIEG has been localized to chromosome 8q22.2 locus, the same locus as the genes involved in osteopetrosis and acute myeloid leukemia and close to the c-myc gene locus and a locus of high polymorphism in cancer biopsies. The correlation between the levels of TIEG protein and the stage of breast cancer, its prime location in human chromosome 8q22.2, and past studies with pancreatic carcinoma, suggests that TIEG may play a role in tumor suppressor gene activities, apoptosis, or some other regulatory function of cell cycle regulation.

Overexpression of the TGFbeta-regulated zinc finger encoding gene, TIEG, induces apoptosis in pancreatic epithelial cells.

Members of the TGFbeta family of peptides exert antiproliferative effects and induce apoptosis in epithelial cell populations. In the exocrine pancreas, these peptides not only regulate normal cell growth, but alterations in these pathways have been associated with neoplastic transformation. Therefore, the identification of molecules that regulate exocrine pancreatic cell proliferation and apoptotic cell death in response to TGFbeta peptides is necessary for a better understanding of normal morphogenesis as well as carcinogenesis of the pancreas. In this study, we have characterized the expression and function in exocrine pancreatic epithelial cells of the TGFbeta-inducible early gene (TIEG), a Krüppel-like zinc finger transcription factor encoding gene previously isolated from mesodermally derived osteoblastic cells. We demonstrate that this gene is expressed in both acinar and ductular epithelial cell populations from the exocrine pancreas. In addition, we show that the expression of TIEG is regulated by TGFbeta1 as an early response gene in pancreatic epithelial cell lines. Moreover, overexpression of TIEG in the TGFbeta-sensitive epithelial cell line PANC1 is sufficient to induce apoptosis. Together, these results support a role for TIEG in linking TGFbeta-mediated signaling cascades to the regulation of pancreatic epithelial cell growth.

Identification of a novel TGF-beta-regulated gene encoding a putative zinc finger protein in human osteoblasts.

The TGF-beta family of growth factors has been extensively studied and found to play major roles in bone physiology and disease. A novel, TGF-beta-inducible early gene (TIEG) in normal human fetal osteoblasts (hFOB) has been identified using differential-display PCR. Using this differentially expressed cDNA fragment of TIEG to screen a hOB cDNA library, a near full-length cDNA for this gene was isolated. Northern analyses indicated that the steady-state levels of the 3.5 kb TIEG mRNA increased within 30 min of TGF-beta treatment of human osteoblasts and reached a maximum of 10-fold above control levels at 120 min post-treatment. This regulation was independent of new protein synthesis. Computer sequence analyses indicates that TIEG mRNA encodes for a 480 amino-acid protein. The TIEG protein contains three zinc finger motifs, several proline-rich src homology-3 (SH3) binding domains at the C-terminal end, and is homologous in this region to the zinc finger-containing transcription factor family of genes. A growth factor/cytokine-specific induction of TIEG has been shown. TIEG expression in hFOB cells was highly induced by TGF-beta and bone morphogenetic protein-2 (BMP-2), with a moderate induction by epidermal growth factor (EGF), but no induction by other growth factors/cytokines was observed. In addition to osteoblastic cells, high levels of TIEG expression were detected in skeletal muscle tissue, while low or no detectable levels were found in brain, lung, liver or kidney. Because TIEG is an early induced putative transcription factor gene, and shows a growth factor induction and tissue specificity, its protein product might play an important role as a signalling molecule in osteoblastic cells.