Transforming growth factor-beta: multifunctional regulator of differentiation and development

Transforming growth factors-beta (TGF-beta) are 25 kilodalton (kDa) homodimeric peptides with multifunctional actions controlling the growth, differentiation and function of a broad range of target cells of both epithelial and mesenchymal derivation. They are expressed early in embryogenesis and their tissue-specific and developmentally dependent expression is strongly suggestive of an essential role in particular morphogenetic and histogenetic events. Five distinct TGF-beta s have been characterized so far, with 65-80% homology to each other. By using both molecular biological and immunohistochemical techniques, we are currently attempting to define specific sites of expression of the different TGF-beta s and to determine whether TGF-beta s 1-5 might have unique functions in development and in the mature organism. Comparative study of the promoter regions for the different TGF-beta s and for any particular TGF-beta in different species is also underway. Mechanistically, TGF-beta s act to control gene expression of their target cells, many of their actions converging on a complex, multifaceted scheme of control of matrix proteins and their interactions with cells; these effects on matrix are thought to mediate many of the effects of TGF-beta on development.     

Transforming growth factor-beta1 as a regulator of the serpins/t-PA axis in cerebral ischemia.

The tissue type plasminogen activator (t-PA) is a serine protease that is involved in neuronal plasticity and cell death induced by excitotoxins and ischemia in the brain. t-PA activity in the central nervous system is regulated through the activation of serine protease inhibitors (serpins) such as the plasminogen activator inhibitor (PAI-1), the protease nexin-1 (PN-1), and neuroserpin (NSP). Recently we demonstrated in vitro that PAI-1 produced by astrocytes mediates the neuroprotective effect of the transforming growth factor-beta1 (TGF-beta1) in NMDA-induced neuronal cell death. To investigate whether serpins may be involved in neuronal cell death after cerebral ischemia, we determined, by using semiquantitative RT-PCR and in situ hybridization, that focal cerebral ischemia in mice induced a dramatic overexpression of PAI-1 without any effect on PN-1, NSP, or t-PA. Then we showed that although the expression of PAI-1 is restricted to astrocytes, PN-1, NSP, and t-PA are expressed in both neurons and astrocytes. Moreover, by using semiquantitative RT-PCR and Western blotting, we observed that only the expression of PAI-1 was modulated by TGF-beta1 treatment via a TGF-beta-inducible element contained in the PAI-1 promoter (CAGA box). Finally, we compared the specificity of TGF-beta1 action with other members of the TGF-beta family by using luciferase reporter genes. These data show that TGF-beta and activin were able to induce the overexpression of PAI-1 in astrocytes, but that bone morphogenetic proteins, glial cell line-derived neutrophic factor, and neurturin did not. These results provide new insights into the regulation of the serpins/t-PA axis and the mechanism by which TGF-beta may be neuroprotective.     

Transforming growth factor-beta1 promotes the morphological and functional differentiation of the myofibroblast

The myofibroblast is responsible for the generation of contractile force associated with wound contraction and pathological contractures and is characterized by the presence of alpha-smooth muscle (alpha-sm) actin-containing stress fibers, vinculin-containing fibronexus adhesion complexes, and fibronectin fibrils containing the ED-A splice variant. Transforming growth factor-beta1 (TGF-beta1) can promote the expression of alpha-sm actin in myofibroblasts, but the functional significance of this increased expression is unclear. In this study, we demonstrate, using the stress-relaxed collagen lattice contraction assay, that TGF-beta1 promoted a dose-dependent increase in the generation of contractile force in myofibroblasts and a concomitant increase in the expression of alpha-sm actin. We also demonstrate that TGF-beta1 enhanced the formation of the structural elements important in myofibroblast contractile force generation and transmission, including stress fibers, vinculin-containing fibronexus adhesion complexes, and fibronectin fibrils, and that this enhancement occurred prior to, and independent of, alpha-sm actin expression. This differentiated myofibroblast phenotype was not stable. Removal of TGF-beta1 resulted in reduced expression of alpha-sm actin as well as a decreased assembly of stress fibers and vinculin-containing adhesion complexes; however, there was no reduction in fibronectin fibrils. We conclude that TGF-beta1 promotes the morphological and functional differentiation of the myofibroblast by first enhancing the formation of the structural elements characteristic of the myofibroblast followed by increased expression of alpha-sm actin and contractile force generation.     

Transforming growth factor-beta pathway: role in pancreas development and pancreatic disease

The pancreas is a complex exocrine and endocrine gland that controls many homeostatic functions. The exocrine pancreas produces and secretes digestive enzymes, whereas, the endocrine pancreas produces four distinct hormones, chief among them being the glucose regulating hormone-insulin. Diabetes, pancreatitis and pancreatic cancer are some of the main afflictions that result from pancreas dysfunction. Transforming growth factor-beta (TGF-beta) proteins are central regulators of pancreas cell function, and have key roles in pancreas development and pancreatic disease. Since expression levels and kinase activities of components of TGF-beta signaling are aberrantly altered in diseases of the pancreas, modulating the activity of TGF-beta provides a unique and rational opportunity for therapeutic intervention. Although TGF-beta still remains elusive in terms of our understanding of its multifunctional modes of action, research is moving closer to the design of approaches directed toward modulating its activities for therapeutic benefit.     

Transforming growth factor-beta 1 (TGF-beta 1) is a bifunctional immune regulator for mucosal IgA responses

Porcine transforming growth factor-beta 1 (TGF-beta 1) exerts a unique bifunctional immunoregulatory effect on IgA responses in murine mesenteric and Peyer's patches lymphocyte cultures. TGF-beta 1 is a potent costimulator of LPS-induced IgA responses. The enhancement was observed at TGF-beta 1 at 0.1 to 10 ng/ml in both early and late phases of IgA responses from Day 5 to Day 14 in cultures. On the contrary, TGF-beta 1 exerts a profound immunosuppressive effect on IL-5-induced IgA synthesis. TGF-beta 1 is a natural cytokine produced by intestinal epithelial cells and may account for the polyclonal production and secretion of IgA at the mucosal surface and ensure the integrity of the primary host defense by mucosa-associated lymphoid tissues (MALT).     

Transforming growth factor-beta(1) regulates differentiation of porcine granulosa cells in vitro

Transforming growth factor-beta (TGF-beta) is a potential regulator of ovarian function and follicular development. It is speculated that TGF-beta mediates the events in the follicle which culminate in ovulation of the oocyte. The complex processes which ultimately leads to this natural phenomenon must involve interactions between the 2 major follicular cell types, theca and granulosa cells, and the oocyte. Furthermore, a complex local regulatory system must exist to determine which follicles should undergo development and, eventually, which of those should ovulate or undergo atresia. To begin to understand this perplexing process, we must first understand the variables which control the function of each individual cell type. This study investigated the effect of TGF-beta(1) on FSH-induced porcine granulosa cell differentiation in vitro. Transforming growth factor-beta(1) was shown to inhibit progesterone production at high concentrations (0.1 and 10.0 ng/ml) after 12-, 24- and 48-hour treatment. However, TGF-beta(1) produced a biphasic effect on FSH-induced progesterone production during the 12-hour interval between the 36- and 48- hour treatment periods; TGF-beta(1) stimulated progesterone production at a low concentration (0.001 ng/ml) and inhibited production at high concentrations (0.1 and 10.0 ng/ml). The results obtained from the biphasic effect were not observed during any of the other incubation periods or intervals investigated. These results show that TGF-beta(1) has opposing effects on the differentiation of porcine granulosa cells as compared with those on rat granulosa cells. Moreover, TGF-beta(1) can produce opposing effects within the porcine granulosa cell itself which are specific to the concentration and treatment period used. The results of this study seem to suggest that TGF-beta(1) is species- and time-specific in its regulatory actions on FSH-induced porcine granulosa cell differentiation.     

Transforming growth factor-beta and Wnt signals regulate chondrocyte differentiation through Twist1 in a stage-specific manner

We investigated the molecular mechanisms underlying the transition between immature and mature chondrocytes downstream of TGF-beta and canonical Wnt signals. We used two developmentally distinct chondrocyte models isolated from the caudal portion of embryonic chick sternum or chick growth plates. Lower sternal chondrocytes exhibited immature phenotypic features, whereas growth plate-extracted cells displayed a hypertrophic phenotype. TGF-beta significantly induced beta-catenin in immature chondrocytes, whereas it repressed it in mature chondrocytes. TGF-beta further enhanced canonical Wnt-mediated transactivation of the Topflash reporter expression in lower sternal chondrocytes. However, it inhibited Topflash activity in a time-dependent manner in growth plate chondrocytes. Our immunoprecipitation experiments showed that TGF-beta induced Sma- and Mad-related protein 3 interaction with T-cell factor 4 in immature chondrocytes, whereas it inhibited this interaction in mature chondrocytes. Similar results were observed by chromatin immunoprecipitation showing that TGF-beta differentially shifts T-cell factor 4 occupancy on the Runx2 promoter in lower sternal chondrocytes vs. growth plate chondrocytes. To further determine the molecular switch between immature and hypertrophic chondrocytes, we assessed the expression and regulation of Twist1 and Runx2 in both cell models upon treatment with TGF-beta and Wnt3a. We show that Runx2 and Twist1 are differentially regulated during chondrocyte maturation. Furthermore, whereas TGF-beta induced Twist1 in mature chondrocytes, it inhibited Runx2 expression in these cells. Opposite effects were observed upon Wnt3a treatment, which predominates over TGF-beta effects on these cells. Finally, overexpression of chick Twist1 in mature chondrocytes dramatically inhibited their hypertrophy. Together, our findings show that Twist1 may be an important regulator of chondrocyte progression toward terminal maturation in response to TGF-beta and canonical Wnt signaling.     

Transforming growth factor-beta 1 expression in irradiated liver

The expression of transforming growth factor-beta 1 (TGF-beta 1) in the liver of irradiated rats was increased in a dose-dependent fashion 9 months after irradiation. Expression of TGF-beta 1 was confined primarily to hepatocytes in the pericentral region of the liver, and the percentage of hepatocytes strongly positive for TGF-beta 1 was significantly correlated with the extent of fibrosis. We further showed that a localized injection of TGF-beta 1 into normal rat liver elicited a strong fibrotic reaction at the injection site. These results suggest that the increased hepatic concentration of TGF-beta 1 in response to radiation injury may be important in the pathogenesis of radiation hepatitis. TGF-beta 1 was also found to be present at a significantly higher concentration in unirradiated human hepatocytes than in normal rat hepatocytes, implying that the propensity for humans to develop radiation hepatitis may result in part from the elevated levels of TGF-beta 1 normally found in human liver.     

Origin of exocrine pancreatic cells from nestin-positive precursors in developing mouse pancreas

During pancreatic development, endocrine and exocrine cell types arise from common precursors in foregut endoderm. However, little information is available regarding regulation of pancreatic epithelial differentiation in specific precursor populations. We show that undifferentiated epithelial precursors in E10.5 mouse pancreas express nestin, an intermediate filament also expressed in neural stem cells. Within developing pancreatic epithelium, nestin is co-expressed with pdx1 and p48, but not ngn3. Epithelial nestin expression is extinguished upon differentiation of endocrine and exocrine cell types, and no nestin-positive epithelial cells are observed by E15.5. In E10.5 dorsal bud explants, activation of EGF signaling results in maintenance of undifferentiated nestin-positive precursors at the expense of differentiated acinar cells, suggesting a precursor/progeny relationship between these cell types. This relationship was confirmed by rigorous lineage tracing studies using nestin regulatory elements to drive Cre-mediated labeling of nestin-positive precursor cells and their progeny. These experiments demonstrate that a nestin promoter/enhancer element containing the second intron of the mouse nestin locus is active in undifferentiated E10.5 pancreatic epithelial cells, and that these nestin-positive precursors contribute to the generation of differentiated acinar cells. As in neural tissue, nestin-positive cells act as epithelial progenitors during pancreatic development, and may be regulated by EGF receptor activity.     

Somatostatin: a potential antigrowth factor for the exocrine pancreas

The increases in DNA synthesis and total DNA content after caerulein treatment support the trophic effect of this CCK analog on the pancreas. Over a 15 day caerulein treatment, pancreatic growth plateaued after 5 days and somatostatin is believed to be responsible for this phenomenon. The present study was undertaken to test this possibility. Rats were treated for 2 or 4 days with caerulein (1 μg · kg^?1), somatostatin antiserum plus caerulein or caerulein plus somatostatin (600 μg · kg^?1). Caerulein increased all parameters studied after 2 and 4 days; pancreatic hyperplasia was established after 2 days. The somatostatin antiserum significantly enhanced the effect of caerulein, especially on DNA synthesis and contents after 2 and 4 days. The trophic effect of caerulein was significantly reduced by somatostatin dramatically so with respect to hyperplasia. The effects of the somatostatin antiserum and those of somatostatin on stimulated pancreatic growth support the hypothesis that somatostatin may be considered an endogenous growth inhibitory factor for the pancreas.     

Transforming growth factor-beta1, transforming growth factor-beta2, and transforming growth factor-beta3 enhance ovarian cancer metastatic potential by inducing a Smad3-dependent epithelial-to-mesenchymal transition

Transforming growth factor-beta (TGF-beta) is thought to play a role in the pathobiological progression of ovarian cancer because this peptide hormone is overexpressed in cancer tissue, plasma, and peritoneal fluid. In the current study, we investigated the role of the TGF-beta/Smad3 pathway in ovarian cancer metastasis by regulation of an epithelial-to-mesenchymal transition. When cancer cells were cultured on plastic, TGF-beta1, TGF-beta2, and TGF-beta3 induced pro-matrix metalloproteinase (MMP) secretion, loss of cell-cell junctions, down-regulation of E-cadherin, up-regulation of N-cadherin, and acquisition of a fibroblastoid phenotype, consistent with an epithelial-to-mesenchymal transition. Furthermore, Smad3 small interfering RNA transfection inhibited TGF-beta-mediated changes to a fibroblastic morphology, but not MMP secretion. When cancer cells were cultured on a three-dimensional collagen matrix, TGF-beta1, TGF-beta2, and TGF-beta3 stimulated both pro-MMP and active MMP secretion and invasion. Smad3 small interfering RNA transfection of cells cultured on a collagen matrix abrogated TGF-beta-stimulated invasion and MMP secretion. Analysis of Smad3 nuclear expression in microarrays of serous benign tumors, borderline tumors, and cystadenocarcinoma revealed that Smad3 expression could be used to distinguish benign and borderline tumors from carcinoma (P = 0.006). Higher Smad3 expression also correlated with poor survival (P = 0.031). Furthermore, a direct relationship exists between Smad3 nuclear expression and expression of the mesenchymal marker N-cadherin in cancer patients (P = 0.0057). Collectively, these results implicate an important role for the TGF-beta/Smad3 pathway in mediating ovarian oncogenesis by enhancing metastatic potential.     

Transforming growth factor-beta 1 binds to immobilized fibronectin

We have characterized the interaction of homodimeric porcine transforming growth factor-beta 1 (TGF-beta 1) with affinity-purified human plasma fibronectin. Using a solid-phase binding assay, we have demonstrated that TGF-beta 1 binds to fibronectin immobilized on Immunlon ITM microtiter plates. TGF-beta 1 binding increased with time, reaching a plateau after 4-6 h, and was dependent upon the concentration of both labeled TGF-beta 1 and immobilized fibronectin present. The binding of radiolabeled TGF-beta 1 to fibronectin was saturable and was reduced 75% in the presence of a 100-fold excess of unlabeled TGF-beta 1. TGF-beta 1 bound to fibronectin with an association rate constant (Ka) of 2.96 x 10(3) M-1 s-1 and did not readily dissociate under various conditions. The binding of TGF-beta 1 to fibronectin was insensitive to variations in ionic strength over a range of 0.1-1.0 M NaCl and was relatively insensitive to divalent cation concentration in the range of 0.1-10.0 mM as well. These data suggest that the binding of TGF-beta 1 to fibronectin may not be dependent upon the interaction of charged amino acids within these two molecules. However, the binding of TGF-beta 1 to fibronectin was strongly pH-dependent and binding decreased dramatically below pH 4.0 and above pH 10.0, suggesting that charged amino acids may influence TGF-beta 1/fibronectin interactions. The association of TGF-beta 1 with immobilized fibronectin or other extracellular matrix components and subsequent dissociation under acidic conditions or by an as-yet-unidentified mechanism may play a role in the distribution and/or activity of this potent growth regulator at sites of tissue injury and inflammation in vivo.     

Transforming growth factor-beta: a neuroprotective factor in cerebral ischemia

Transforming growth factor-β (TGF-β) has diverse and multiple roles throughout the body. This review focuses on the evidence supporting its functions in the central nervous system, with a particular emphasis on its purported role in cerebral ischemia. Numerous studies have documented that TGF-β1 levels are enhanced in the brain following cerebral ischemia. As evidence that such an upregulation is beneficial, agonist studies have demonstrated that TGF-β1 reduces neuronal cell death and infarct size following middle cerebral artery occlusion (MCAO), while conversely, antagonist studies have shown increased neuronal cell death and infarct size after MCAO. These studies suggest that TGF-β1 has a neuroprotective role in cerebral ischemia. Recent work with adenoviral-mediated overexpression of TGF-β1 in vivo in mice has further implicated a neuroprotective role for TGF-β1 in cerebral ischemia, as evidenced by a reduction in neuronal cell death, infarct size, and neurological outcome. Additionally, numerous in vitro studies have documented the neuroprotective ability of TGF-β1 in neurons from a variety of species, including rats, mice, chicks, and humans. Of significant interest, TGF-β1 was shown to be protective against a wide variety of death-inducing agents/insults, including hypoxia/ischemia, glutamate excitotoxicity, β-amyloid, oxidative damage, and human immunodeficiency virus. The mechanism of TGF-β1-mediated neuroprotection remains to be resolved, but early evidence suggests that TGF-β1 regulates the expression and ratio of apoptotic (Bad) and antiapoptotic proteins (Bcl-2, Bcl-x₁), creating an environment favorable for cell survival of death-inducing insults. Taken as a whole, these results suggest that TGF-β1 is an important neuroprotective factor that can reduce damage from a wide-array of death-inducing agents/insults in vitro, as well as exert protection of the brain during cerebral ischemia. The authors’ research is supported by research grants (HD-28964 and AG-17186 to DWB) from the National Institutes of Health, NICHD, and NIA.     

Transforming growth factor-beta receptor-associated protein 1 is a Smad4 chaperone

Members of the transforming growth factor-beta (TGF-beta) superfamily signal through unique cell membrane receptor serine-threonine kinases to activate downstream targets. TRAP1 is a previously described 96-kDa cytoplasmic protein shown to bind to TGF-beta receptors and suggested to play a role in TGF-beta signaling. We now fully characterize the binding properties of TRAP1, and show that it associates strongly with inactive heteromeric TGF-beta and activin receptor complexes and is released upon activation of signaling. Moreover, we demonstrate that TRAP1 plays a role in the Smad-mediated signal transduction pathway, interacting with the common mediator, Smad4, in a ligand-dependent fashion. While TRAP1 has only a small stimulatory effect on TGF-beta signaling in functional assays, deletion constructs of TRAP1 inhibit TGF-beta signaling and diminish the interaction of Smad4 with Smad2. These are the first data to identify a specific molecular chaperone for Smad4, suggesting a model in which TRAP1 brings Smad4 into the vicinity of the receptor complex and facilitates its transfer to the receptor-activated Smad proteins.     

Transforming growth factor-beta in T-cell biology

Strict control of T-cell homeostasis is required to permit normal immune responses and prevent undesirable self-targeted responses. Transforming growth factor-beta (TGF-beta) has been shown to have an essential role in that regulation. Owing to its broad expression, and inhibitory effects on multiple cell types of the immune system, TGF-beta regulation is complex. Through advances in cell-specific targeting of TGF-beta signalling in vivo, the role of TGF-beta in T-cell regulation has become clearer. Recent in vitro studies provide a better understanding of how TGF-beta regulates T-cell homeostasis, through multiple mechanisms involving numerous cell types.     

Transforming growth factor-beta 1 induces expression of statin during differentiation of human promonocytic leukemia cells.

Transforming growth factor-Beta (TGF-beta) is a potent growth inhibitor for several cell types including epithelial cells and hematopoietic progenitor cells. Using a human promonocytic leukemia cell line, THP-1, we have shown that TGF-beta inhibits their proliferation and promotes differentiation into cells exhibiting macrophage-like properties. Therefore, a key question is whether TGF-beta influences the expression of genes associated with proliferation and/or growth inhibition. TGF-beta treatment of THP-1 cells results in downregulation of expression of c-myc. We also observe that TGF-beta 1-treated cells express reduced levels of the cell cycle regulated histone, H2B, but express elevated levels of an RNA splicing variant of this histone that has been observed to be upregulated in growth inhibited and terminally differentiated cells. In addition, a nuclear protein associated with senescence and withdrawal of cells from the cell cycle, statin, is also expressed by THP-1 cells in response to TGF-beta 1 treatment. These results suggest that TGF-beta 1 is capable of inducing expression of specific nuclear proteins associated with differentiation and/or cessation of proliferation that may result in changes in nuclear organization and altered gene expression. Such changes in nuclear organization may be incompatible with continued proliferation of the cells.