Expression of Recombinant Extracellular Domain of the Type II Transforming Growth Factor-β Receptor: Utilization in a Modified Enzyme-Linked Immunoabsorbent Assay to Screen TGF-β Agonists and Antagonists

TGF-β is a ubiquitous protein that exhibits a broad spectrum of biological activity. The prokaryotic expression and purification of the extracellular domain of the type II TGF-β receptor (TβR-II-ED), without the need for fusion protein cleavage and refolding, is described. The recombinant TβR-II-ED fusion protein bound commercially available TGF-β1 and displayed an affinity of 11.1 nM. In a modified ELISA, receptor binding to TGF-β1 was inhibited by TGF-β3. The technique lends itself to high-throughput screening of combinatorial libraries for the identification of TGF-β agonists and antagonists and this, in turn, may have important therapeutic implications.     

Modulation of gene expression in the preimplantation mouse embryo by TGF-α and TGF-β

The effect of growth factors on regulating gene expression in the preimplantation mouse embryo was examined, since results of previous experiments revealed a stimulatory effect of exogenously-added growth factors on preimplantation development in vitro. Treatment of early cavitating blastocysts with either 250 pM TGF-α or TGF-β results in changes in the pattern of total protein synthesis as assessed by high-resolution two-dimensional gel electrophoresis. In some cases, the synthesis of a particular polypeptide is either up- or downregulated by each growth factor, whereas in other instances the synthesis of a polypeptide is modulated by one but not the other growth factor. Use of the mRNA differential display method permitted the identification of genes whose expression is either up- or downregulated by these growth factors. Treatment of mouse blastocysts with either TGF-α or TGF-β results in the increased expression of the b subunit of the F₀ ATPase. TGF-β also stimulates the expression of the DNA polymerase α. TGF-α treatment results in the increase in expression of a gene homologous to the human HEPG2 cDNA, as well as in a decrease in expression of fibronectin. © 1995 Wiley-Liss, Inc.     

Localization of Transforming Growth Factor-β Type I and Type II Receptors in Mouse Development

We have investigated the localization pattern of the transforming growth factor-β (TGF-β) receptors type I (TβR-I) and type II (TβR-II) during mouse organogenesis by immunohistochemical analysis. Staining of both receptors was found in many developing organs, e.g., bone, teeth, Meckel's cartilage, and neural tissues, where the expression of their ligands has been previously reported. During the investigated stages, expression of TβR-I was more ubiquitous than that of TβR-II. TβR-II preferentially localized in the undifferentiated mesenchymal cells which subsequently differentiated into bone. There was no staining of TβR-II in the central nervous system, while intense TβR-I staining was found specifically in nervous tissues. Expression of TβR-I and TβR-II was mostly coincident with that of their ligands, suggesting that TGF-βs act as multiple mediators during organogenesis. In addition, colocalization of both receptors in the epithelia of the tooth bud and submandibular gland, which were actively invaginating into the mesenchyme, leads us to speculate that both receptors may be necessary for dynamic epithelial morphogenesis.     

TGF-β: 20 years and counting

The history of transforming growth factor-beta (TGF-β) as a bifunctional agent in the immune system is briefly described. The importance of cellular context in understanding the role of TGF-β in regulating immune response is emphasized.     

TGF-β and cancer

The relationships between transforming growth factor-β (TGF-β) and cancer are varied and complex. The paradigm that is emerging from the experimental evidence accumulated over the past decade or so is that TGF-β can play two different and opposite roles with respect to the process of malignant progression. During early stages of carcinogenesis, TGF-β acts predominantly as a potent tumor suppressor and may mediate the actions of chemopreventive agents such as retinoids and nonsteroidal anti-estrogens. However, at some point during the development and progression of malignant neoplasms, bioactive TGF-βs make their appearance in the tumor microenvironment and the tumor cells escape from TGF-β-dependent growth arrest. In many cases, this resistance to TGF-β is the consequence of loss or mutational inactivation of the genes that encode signaling intermediates. These include the types I and II TGF-β receptors, as well as receptor-associated and common-mediator Smads. The stage of tumor development or progression at which TGF-β-resistant clones come to dominate the tumor cell population in different types of neoplasm remains to be defined. The phenotypic switch from TGF-β-sensitivity to TGF-β-resistance that occurs during carcinogenesis has several important implications for cancer prevention and treatment.     

TGF-β: from latent to active

The transforming growth factor-betas (TGF-βs) are synthesized as precursor proteins that are modified intracellularly prior to secretion. One of the most relevant intracellular modifications is the cleavage of the C-terminal pro-region from the N-terminal portion of the protein. The C-terminal pro-region is referred to as the latency-associated peptide (LAP) while the N-terminal region is called the mature TGF-β or active TGF-β. However, with some exceptions the LAP noncovalently associates with the mature TGF-β prior to secretion. When the mature TGF-β is associated with the LAP it is called L-TGF-β and cannot interact with its receptor and has no biological effect. The TGF-βs and their receptors are very ubiquitously expressed, suggesting that the regulation of TGF-β activity is likely to be complex and multifactorial. However, one of the most important means of controlling the biological effects of TGF-β is the regulation of converting L-TGF-β to active TGF-β. The current literature supports two major mechanisms of activation of L-TGF-β and suggests that the mechanism of activation of L-TGF-β may be varied and context-dependent. For TGF-β to become biologically active the LAP has to be either released from its associations with L-TGF-β or undergo conformational change such that the LAP is not released from the L-TGF-β complex but exposes the TGF-β receptor binding site. Since TGF-β has been associated with the pathogenesis of numerous diseases, the various mechanisms of activation of L-TGF-β in context offer the possibility of controlling TGF-β activity localized to the organ of involvement and to a more specific disease process.     

Structure,Function, and Regulation of the Three β-Adrenergic Receptors

Three β-adrenergic receptor subtypes are now known to be functionally expressed in mammals. All three belong to the R₇G family of receptors coupled to G-proteins, and characterized by an extracellular glycosylated N-terminal and an intracellular C-terminal region and seven transmembrane domains, linked by three exta- and three intracellular loops. The catecholamine ligand binding domain, studied using affinity-labeling and site-directed mutagenesis, is a pocket lined by residues belonging to the transmembrane domains. The region responsible for the interaction with the Gs protein which, when activated, stimulates adenylyl cyclase, is composed of residues belonging to the parts most proximal to the membrane of intracellular loop i₃ and the C-terminal region. The pharmacology of the three subtypes is quite distinct: in fact most of the potent β₁/β₂ antagonists (the well known β blockers) act as agonists on β₃. The subtype is resistant to short-term desensitization mediated by phosphorylation through PKA or βARK, in stark contrast to the β₁ or β₂ subtypes. Various compounds (dexamethasone, butyrate, insulin) up regulate β₁ or β₁ subtypes while down-regulating β₃ whose expression strictly correlates with differentiation of 3T3-F442A fibroblasts into adipocytes, thus confirming that the expression of the three subtypes may each be regulated independently to exert a specific physiologic role in different tissues or at different stages of development.     

TGF-β in infections and infectious diseases

Since it was first described as having the ability to inhibit macrophage activation, transforming growth factor-beta (TGF-β) has been analyzed for its role in regulating immune responses to a variety of pathogens, including viruses, bacteria, yeast, and protozoa. Most of the studies have involved organisms that infect macrophages, and this discussion will attempt to highlight these findings. Perhaps the most work has been performed with protozoan pathogens, including Trypanosoma cruzi and a variety of Leishmania species, so the discussion will begin with these organisms. Other studies have focused on mycobacteria and viruses, including human immunodeficiency virus, so these areas will also be emphasized in the discussion. For the most part, investigators have reported that TGF-β has, as expected, a negative influence on host responses and a beneficial effect on the survival and growth of intracellular pathogens. However, other studies have found that TGF-β may have a positive or beneficial effect in some models of infection. This review will attempt to highlight studies and conclusions on the roles of TGF-β in infection.     

Regulation of GM-CSF-Induced Dendritic Cell Development by TGF-β1 and Co-Developing Macrophages

Using a culture system of bone marrow progenitor cells with GM-CSF and TGF-β1, a study was performed to analyze the effect of TGF-β1 on the development of dendritic cells (DC) and to elucidate the regulatory role of macrophages co-developing with dendritic cells. The results demonstrate that DC generated in the presence of TGF-β1 were immature with respect to the expression of CD86, nonspecific esterase activity and cell shape. Such inhibitory effects of TGF-β1 were dependent on FcR⁺ macrophages, which were depleted by panning. TGF-β1 did not appear to inhibit the commitment of progenitor cells to the DC lineage. In addition, TGF-β1 also acted directly on the intermediate stage of DC to prevent their over-maturation, which results in a preferential decrease in MHC class II, but not in CD86, in the presence of TNF-α. FcR⁺ suppressive macrophages were also shown to facilitate DC maturation when stimulated via FcR-mediated signals even in the presence of TGF-β1. These results indicate that TGF-β1 indirectly and directly regulate the development of DC and that co-developing macrophages have a regulatory role in DC maturation.     

Transforming growth factor-β enhances secretory component and major histocompatibility complex class I antigen expression on rat IEC-6 intestinal epithelial cells

Transforming growth factor-β (TGF-β) has been implicated as having a role in inflammatory responses by inducing cellular infiltration and the release of inflammatory cytokines. In this study, the IEC-6 rat intestinal epithelial cell line was used as a model to assess the effect of TGF-β₁ on the expression of various plasma membrane determinants. TGF-β₁ induced a dose-dependent increase in the percentage of cells expressing surface secretory component (SC) and class I major histocompatibility (MHC) antigens. However, the expression of class II MHC was unaffected. In contrast, epidermal growth factor had no effect on any of the surface proteins studied. The TGF-β₁-enhanced expression of SC was accompanied by an enhanced binding of polymeric, but not monomeric, immunoglobulin A (IgA). Preincubation of the TGF-β₁-treated cells with an anti-human β-galactosyltransferase (β-GT) antiserum did not block the binding of the anti-SC antibody, indicating that the TGF-β-induced increase in SC staining was due to SC expression and not the polymeric immunoglobulin-binding enzyme, β-GT. These results indicate that TGF-β₁ may be important in immune functions involving intestinal epithelial cells by enhancing the expression of surface class I MHC antigens and SC, a protein responsible for the transport of polymeric IgA into the intestinal lumen.     

Correlation between TGF-β2/3 promoter DNA methylation and Smad signaling during palatal fusion induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a persistent organic pollutant that is strongly associated with a number of human diseases and birth defects, including cleft palate. Transforming growth factor (TGF) plays a significant role during mammalian palatogenesis. However, the epigenetic mechanism of transforming growth factors in the process of TCDD-induced cleft palate is unclear. The purpose of this research was to investigate the relationship and potential mechanism between TGF-β2/3 promoter DNA methylation and Smad signaling during TCDD-induced cleft palate. Pregnant C57BL/6N mice were exposed to 64 µg/kg TCDD on gestational day 10 (GD10) to establish the cleft palate model and palatal tissues of embryos were collected on GD13, GD14, and GD15 for subsequent experiments. TGF-β2/3 mRNA expression, TGF-β2/3 promoter methylation, and Smad signaling molecules expression were assessed in the palate of the two groups. The results showed that the incidence of cleft palate was 94.7% in the TCDD-treated group whereas no cleft palate was found in the control group. TCDD-treated group altered specific CpG sites of TGF-β2/3 promoter methylation. Compared to the control group, the proliferation of mouse embryonic palate mesenchymal stromal cells (MEPM), the expressions of TGF-β2/3, p-Smad2, and Smad4 were all reduced, while the expression of Smad7 was significantly increased in the atAR group. Smad signaling was downregulated by TCDD. Therefore, we suggest that TGF-β2/3 promoter methylation and Smad signaling may be involved in TCDD-induced cleft palate formation in fetal mice.     

Terminal Differentiation of Normal Human Oral Keratinocytes Is Associated with Enhanced Cellular TGF-β and Phospholipase C-γ1 Levels and Apoptotic Cell Death

Subculture of primary normal human oral keratinocytes (NHOK) results in terminal differentiation, leading to cell death. To investigate whether the subculture-induced death of NHOK is due to apoptosis, we studied transferase-mediated dUTP nick end labeling (TUNEL)-positive cells, DNA fragmentation, and expression of several apoptosis-associated genes from NHOK with different passage numbers. We also determined the effect of transforming growth factor β₁ (TGF-β₁) on the induction of apoptosis in NHOK. We were able to subculture primary NHOK up to the fifth passage, at which point cells showed morphological features of differentiation. Appearance of DNA fragmentation concurrently occurred with an increase in the number of TUNEL-positive cells with higher passage numbers. The level of cellular p53 proteins was gradually decreased by the continued passage of cells, whereas the levels of intracellular and secreted TGF-β and phospholipase C-γ1 (PLC-γ1) were significantly elevated by serial subculture. Exogenous TGF-β₁ also induced differentiation and apoptosis of proliferating NHOK. These data indicate that terminal differentiation of NHOK is associated with apoptosis, which is, in part, linked to elevated cellular levels of TGF-β and PLC-γ1.     

TGF-β1: immunosuppressant and viability factor for T lymphocytes

Transforming growth factor-beta (TGF-β) is a multifunctional cytokine with multiple roles in the immune system. To date, it has been difficult to develop a comprehensive picture of the effect of TGF-β on T lymphocytes, because TGF-β not only acts directly on T lymphocytes, but also acts indirectly by regulating the function of antigen-presenting cells. In early studies, it was mostly the inhibitory function of TGF-β that was demonstrated; recently, however TGF-β was recognized as an antiapoptotic survival factor for T lymphocytes. The outcome of the TGF-β effect on T lymphocytes was shown to strongly depend on their stage of differentiation and on the cytokine milieu. TGF-β cannot be classified as a classical Th1 or Th2 cytokine. However, recently the existence of the TGF-β-producing Th3 subset was described which might play an important regulatory role during an immune response. A better understanding of the molecular mechanism of how TGF-β inhibits or stimulates T lymphocytes will help to predict the complex functions of this cytokine.