Differential expression and biological activity of retinoic acid–induced TGFβ isoforms in embryonic palate mesenchymal cells

The effect of retinoic acid (RA) on TGF-β mRNA expression and protein production in murine embryonic palate mesenchymal (MEPM) cells was examined by Northern blotting and TGF-β bioassay in association with TGF-β isoform-specific neutralizing antibodies. Heat or acid activation was used to distinguish between latent and active TGF-β protein released into the culture medium. RA had little or no effect on TGF-β1 mRNA expression and protein production. In contrast, RA increased TGF-β2 and β3 protein released into the culture medium, the protein being mostly in an inactive or latent form. The amount of active TGF-β released was increased relative to the total increase in TGF-β released, suggesting that RA treatment stimulated activation of latent TGF-β. RA also increased TGF-β2 mRNA expression; we have previously shown that RA upregulates TGF-β3 mRNA in these cells. RA and TGF-β individually inhibited ³H-thymidine incorporation into MEPM cell DNA, while, when administered simultaneously, they inhibited proliferative activity to a greater extent. Heat- or acid-activated conditioned medium (CM) from MEPM cells treated with RA was able to inhibit ³H-thymidine incorporation into MEPM cell DNA to an extent greater than seen with RA treatment alone. Coincubation of heat-activated CM from RA-treated MEPM cells with pan-specific or TGF-β2 or β3-specific neutralizing antibodies partially relieved the inhibitory effect on ³H-thymidine incorporation, suggesting that this proliferative response was due to RA-induced TGF-β. Simultaneous treatment with RA and TGF-β also stimulated gycosaminoglycan (GAG) synthesis to an extent greater than that seen with TGF-β treatment alone, this despite the ability of RA to inhibit GAG synthesis. These data demonstrate a role for RA and RA-induced TGF-β in the regulation of palate cell proliferation and GAG synthesis and suggest a role for TGF-β in retinoid-induced cleft palate. J. Cell. Physiol. 177:36–46, 1998. © 1998 Wiley-Liss, Inc.     

TGF-β signalling is mediated by two autonomously functioning TβRI:TβRII pairs

Transforming growth factor (TGF)-βs are dimeric polypeptides that have vital roles in regulating cell growth and differentiation. They signal by assembling a receptor heterotetramer composed of two TβRI:TβRII heterodimers. To investigate whether the two heterodimers bind and signal autonomously, one of the TGF-β protomers was substituted to block receptor binding. The substituted dimer, TGF-β3 WD, bound the TβRII extracellular domain and recruited the TβRI with affinities indistinguishable from TGF-β3, but with one-half the stoichiometry. TGF-β3 WD was further shown to retain one-quarter to one-half the signalling activity of TGF-β3 in three established assays for TGF-β function. Single-molecule fluorescence imaging with GFP-tagged receptors demonstrated a measurable increase in the proportion of TβRI and TβRII dimers upon treatment with TGF-β3, but not with TGF-β3 WD. These results provide evidence that the two TβRI:TβRII heterodimers bind and signal in an autonomous manner. They further underscore how the TGF-βs diverged from the bone morphogenetic proteins, the ancestral ligands of the TGF-β superfamily that signal through a RI:RII:RII heterotrimer.     

Interleukin-1β suppresses apoptosis in CD34 positive bone marrow cells through activation of the type I IL-1 receptor

Interleukin-1 is a pleiotropic cytokine that has been shown previously to suppress active cell death in T cells. Two cell surface receptors for interleukin-1 have been identified and their genes cloned, type I (IL-RI) and type II (IL-RII) receptors. In the present study, we provide evidence for a role of interleukin-1β in the short-term suppression of cell death both in purified CD34⁺/Lin⁻ bone marrow precursors and in the GM-CSF dependent cell line TF-1. Several lines of evidence suggest that the biologic effects of IL-1β are mediated by activation of type I IL-1 receptors (IL-1RI) and induction of GM-CSF production. First, neutralizing antibodies to IL-1RI but not IL-1RII drastically abrogated cell survival induced by IL-1β in CD34⁺/Lin⁻ cells and TF-1 cells. Second, neutralizing antibodies against GM-CSF abrogate cell survival supported by IL-1 both in CD34⁺/Lin⁻ bone marrow cells and in the cell line TF-1. Furthermore, exposure of TF-1 cells to IL-1β results in a transient accumulation of GM-CSF mRNA, with a peak at 3 h, which was dramatically decreased by neutralizing anti-IL-1RI antibodies. In contrast, neutralizing anti-IL-1RII did not change the IL-1 induced cell survival of bone marrow cells and was followed by a paradoxical increase in viable cell numbers, in c-myc and c-myb mRNA accumulation in IL-1 treated TF-1 cells. Together our results indicate that the increase in cell survival induced IL-1β occurs through binding to IL-1RI and the subsequent production of endogenous GM-CSF. IL-1RII does not appear to be involved in signal transduction in primary CD34⁺/Lin⁻ cells but could negatively regulate the response to IL-1β in TF-1 cells. © 1996 Wiley-Liss, Inc.     

Upregulation of transforming growth factor-beta type I receptor by interferon consensus sequence-binding protein in osteosarcoma cells

Transforming growth factor-beta (TGF-β) is a known tumor suppressor, which also exerts a tumor promoting activity at an advanced stage of cancer. Previously, we reported that expression of interferon consensus sequence-binding protein (ICSBP), also known as interferon regulatory factor-8, is positively correlated with TGF-β type I receptor (TGF-β RI) expression in osteosarcoma patient tissues. In this study, we demonstrated that ICSBP upregulated TGF-β RI and induced epithelial-to-mesenchymal transition-like phenomena in human osteosarcoma cell lines. As determined by soft agar growth of osteosarcoma cells and xenografted mouse models, ICSBP increased tumorigenicity, which was reversed by ICSBP knock-down or a TGF-β RI inhibitor. To test whether ICSBP directly regulates the promoter activity of TGF-β RI, we performed a TGF-β RI promoter assay, an electro mobility shift assay, and a chromatin immunoprecipitation assay. We observed that TGF-β RI promoter was activated in ICSBP-overexpressing osteosarcoma cells. Exploiting serial deletions and mutations of the TGF-β RI promoter, we found a putative ICSBP-binding site at nucleotides −216/−211 (GGXXTC) in the TGF-β RI promoter. Our data suggest that ICSBP upregulates TGF-β RI expression by binding to this site, causing ICSBP-mediated tumor progression in osteosarcoma cells. In addition, we found a positive correlation between ICSBP and TGF-β RI expression in several types of tumors using the cBioportal database.SummaryWe demonstrated that interferon consensus sequence-binding protein upregulates transforming growth factor-beta type I receptor (TGF-β RI) expression by binding to nucleotides −216/−211 (GGXXTC) in the TGF-β RI promoter, which resulted in increased tumorigenicity and tumor progression in human osteosarcoma cells.     

Transforming growth factor β1 induces mitogenesis in fetal rat brown adipocytes

The presence of transforming growth factor β₁ (TGF-β₁) for 24 or 48 h stimulated DNA synthesis, the percentage of cells in the S + G₂/M phases of the cell cycle, and cell number, as compared to quiescent cells. The mitogenic capacity of TGF-β₁ (1 pM) was similar to that shown by 10% fetal calf serum (FCS). TGF-β₁ for 48 h increased by 5-fold the percentage of cells containing (³H)thymidine-labeled nuclei as compared to quiescent cells. In addition, single fetal brown adipocytes, showing their typical multilocular fat droplets phenotype, become positive for (³H)thymidine-labeled nuclei in response to TGF-β₁. Moreover, TGF-β₁ induced the mRNA expression of a complete set of proliferation-related genes, such as c-fos (30 min), c-myc and β-actin (2 h), and H-ras, cdc2 kinase, and glucose 6-phosphate dehydrogenase (G6PD) at 4 and 8 h, as compared to quiescent cells. Concurrently, TGF-β₁ for 12 h increased the protein content of proliferating cellular nuclear antigen (PCNA) by 6-fold and p21-ras by 2-fold. Although our results demonstrate that TGF-β₁ induces the expression of very early genes related to cell proliferation, TGF-β₁ could be acting either as a mitogen or as a survival factor to induce proliferation in fetal brown adipocytes. © 1996 Wiley-Liss, Inc.     

Role for autocrine TGF-β1 in regulating differentiation of a human leukemic cell line toward osteoclast-like cells

Increasing evidence suggests that transforming growth factor-β (TGF-β) is involved in bone formation during remodeling. Using a recently cloned human leukemic cell line (FLG 29.1 cells) we demonstrate that these cells synthesize and secrete TGF-β1 and that exogenous or autocrine TGF-β1 can induce the same features of osteoclastic-like cells, exerting its effects through the binding to TGF-β specific receptors. Scatchard analysis of ¹²⁵I-labeled TGF-β1 to FLG 29.1 cells revealed the presence of a single high affinity binding site with a Kd value of ～25 pM and a binding capacity of ～900 sites/cell. Affinity labeling experiments showed that FLG 29.1 cells express type I and type II TGF-β receptors. Stimulation of FLG 29.1 cells with low TGF-β1 doses reduced cell proliferation and increased cell adhesion and tartrate resistant acid phosphatase (TRAcP) activity. Pretreatment of FLG 29.1 cells with TGF-β1 caused a significant and dose-dependent response to calcitonin. Northern blot of total mRNA and analysis of the conditioned media (CM) showed that TGF-β1 was synthesized by FLG 29.1 cells. TPA treatment, which induces partial differentiation of these cells, markedly increased TGF-β1 mRNA expression and growth factor release. The majority of TGF-β1 secreted by TPA-treated cells was in its latent form. However, anti-TGF-β antibodies inhibited TGF-β1 and TPA-induced growth inhibition, calcitonin responsiveness, and TRAcP activity, suggesting that the TPA effect is mediated in part by autocrine TGF-β1 and indicating that the cells can activate and respond to the TGF-β that they secrete. These findings support a potential autocrine role for TGF-β1 in osteoclast differentiation. © 1994 Wiley-Liss, Inc.     

Transforming growth factor-β signaling: Tumorigenesis and targeting for cancer therapy

Transforming growth factor (TGF)-β is a multitasking cytokine such that its aberrant expression is related to cancer progression and metastasis. TGF-β is produced by a variety of cells within the tumor microenvironment (TME), and it is responsible for regulation of the activity of cells within this milieu. TGF-β is a main inducer of epithelial–mesenchymal transition (EMT), immune evasion, and metastasis during cancer progression. TGF-β exerts most of its functions by acting on TβRI and TβRII receptors in canonical (Smad-dependent) or noncanonical (Smad-independent) pathways. Members of mitogen-activated protein kinase, phosphatidylinositol 3-kinase/protein kinase B, and nuclear factor κβ are involved in the non-Smad TGF-β pathway. TGF-β acts by complex signaling, and deletion in one of the effectors in this pathway may influence the outcome in a diverse way by taking even an antitumor role. The stage and the type of tumor (contextual cues from cancer cells and/or the TME) and the concentration of TGF-β are other important factors determining the fate of cancer (progression or repression). There are a number of ways for targeting TGF-β signaling in cancer, among them the special focus is on TβRII suppression.     

Peptide ligands that use a novel binding site to target both TGF-β receptors

The transforming growth factor beta (TGF-β) signaling pathway plays myriad roles in development and disease. TGF-β isoforms initiate signaling by organizing their cell surface receptors TβRI and TβRII. Exploration and exploitation of the versatility of TGF-β signaling requires an enhanced understanding of structure-function relationships in this pathway. To this end, small molecule, peptide, and antibody effectors that bind key signaling components would serve as valuable probes. We focused on the extracellular domain of TβR1 (TβRI-ED) as a target for effector screening. The observation that TβRI-ED can bind to a TGF-β coreceptor (endoglin) suggests that the TβRI-ED may have multiple interaction sites. Using phage display, we identified two peptides LTGKNFPMFHRN (Pep1) and MHRMPSFLPTTL (Pep2) that bind the TβRI-ED (K(d)≈ 10(-5) M). Although our screen focused on TβRI-ED, the hit peptides interact with the TβRII-ED with similar affinities. The peptide ligands occupy the same binding sites on TβRI and TβRII, as demonstrated by their ability to compete with each other for receptor binding. Moreover, neither interferes with TGF-β binding. These results indicate that both TβRI and TβRII possess hot spots for protein-protein interactions that are distinct from those used by their known ligand TGF-β. To convert these compounds into high affinity probes, we exploited the observation that TβRI and TβRII exist as dimers on the cell surface; therefore, we assembled a multivalent ligand. Specifically, we displayed one of our receptor-binding peptides on a dendrimer scaffold. We anticipate that the potent multivalent ligand that resulted can be used to probe the role of receptor assembly in TGF-β function.     

Interleukin-1 binding and prostaglandin E2 synthesis by amnion cells in culture: regulation by tumor necrosis factor-α, transforming growth factor-β, and interleukin-1 receptor antagonist

Proinflammatory cytokines may promote preterm labor in the setting of intrauterine infection. Tumor necrosis factor (TNF) and interleukin-1 (IL-1) synergistically stimulate the production of prostaglandin E₂ (PGE₂) by amnion cells. Transforming growth factor-β (TGF-β) inhibits the cytokine-stimulated PGE₂ production. In the present study, we investigated the binding of IL-1β on human amnion cells in culture. Untreated amnion cells possessed 540±60 IL-1 receptors per cell, with a dissociation constant of 1.4±0.4 nM. Cells treated with TGF-β1 (10 ng/ml) had 570±110 receptors per cell. TNF-α (50 ng/ml) increased the number of IL-1 receptors to 2930±590. TGF-β1 inhibited the receptor upregulation by TNF-α. Cells treated with TGF-β1 and TNF-α expressed 1140±590 receptors per cell. The binding affinity was not changed by the cytokines. IL-1 receptor antagonist (IL-1ra) inhibited the stimulation of amnion cell PGE₂ production by IL-1β, but not by TNF-α. Amnion cells secreted large amounts of IL-1ra (1.1±0.3 ng/10⁵ cells). Treatment of the cells with TGF-β1 or TNF-α did not affect the release of IL-1ra. We conclude that IL-1 receptor expression is an important step in the regulation of the effects of cytokines on amnion cell PGE₂ production.     

Retinoids potentiate transforming growth factor-β activity in bovine endothelial cells through up-regulating the expression of transforming growth factor-β receptors

Retinoic acid (RA) induces the activation of latent transforming growth factor-β (TGF-β) in bovine aortic endothelial cells (BAECs) via enhancement of cellular plasminogen activator (PA)/plasmin levels. The resultant TGF-β suppresses the excessive fibrinolytic activity by decreasing PA expression and stimulating expression of the PA inhibitor, PA inhibitor-1 (PAI-1), and inhibits cell proliferation. Here, we report that, in this regulatory system, RA simultaneously up-regulates the expression of TGF-β receptor types I and II, resulting in enhancement of TGF-β activity in the cells. RA increased the numbers of high- and low-affinity binding sites for ¹²⁵I-TGF-β1 2.1-fold and 1.5-fold, respectively, without alteration of their Kd values. Affinity labeling and Western and Northern blotting studies showed that, following RA treatment, surface levels of both type I and type II receptors increased due to augmentation in their mRNA levels. The effect was dose- and time-dependent. Treatment with 1 μM RA for 15 hr increased mRNA levels of type I and II receptor threefold and eightfold, respectively. Pretreatment of BAECs with either RA or retinol lowered the concentration of TGF-β1 required to suppress PA levels, to enhance PAI-1 levels, and to inhibit cell proliferation. Thus, retinoids may regulate cellular functions of BAECs not only by inducing the formation of active TGF-β but also by stimulating TGF-β receptor expression. This regulatory mechanism may sustain TGF-β-mediated regulation of EC function at a focal site where RA is acting. J. Cell. Physiol. 176:565–573, 1998. © 1998 Wiley-Liss, Inc.     

Single-molecule imaging revealed enhanced dimerization of transforming growth factor β type II receptors in hypertrophic cardiomyocytes

Transforming growth factor β (TGF-β) signaling plays an important role in the pathogenesis of cardiac hypertrophy. However, the molecular mechanism of TGF-β signaling during the process of cardiac remodeling remains poorly understood. In the present study, by employing single-molecule fluorescence imaging approach, we demonstrated that in neonatal rat cardiomyocytes, TGF-β type II receptors (TβRII) existed as monomers at the low expression level, and dimerized upon TGF-β1 stimulation. Importantly, for the first time, we found the increased dimerization of TβRII in hypertrophic cardiomyocytes comparing to the normal cardiomyocytes. The enhanced TβRII dimerization was correlated with the enhanced Smad3 phosphorylation levels. These results provide new information on the mechanism of TGF-β signaling in cardiac remodeling.     

Distinctive mechanism for sustained TGF-β signaling and growth inhibition: MEK1 activation-dependent stabilization of type II TGF-β receptors

There are multiple mechanisms by which cells evade TGF-β-mediated growth inhibitory effects. In this report, we describe a novel mechanism by which cells become resistant to TGF-β-mediated growth suppression. Although having all the components of the TGF-β signaling pathway, different cell lines, RL, HaCaT, and BJAB, have different sensitivities toward TGF-β-induced growth suppression. The TGF-β resistance of RL, a B-cell lymphoma cell line, was due to ligand-induced downregulation of TGF-β receptor II (TβRII) and only transient TGF-β induced nuclear translocation of Smad2 and Smad3. With low-dose phorbol 12-myristate 13-acetate (PMA) or anti-IgM treatment, TGF-β sensitivity was restored by stabilizing TβRII expression and sustaining TGF-β signaling. The MEK inhibitor, U0126, blocked both PMA- and anti-IgM-induced upregulation of TβRII. In HaCaT and BJAB, two TGF-β-sensitive cell lines, which had higher basal levels of phospho-MEK and TβRII compared with RL, U0126 induced downregulation of TβRII and blocked subsequent TGF-β signaling. Similar results were also obtained with normal B cells, where MEK1 inhibitor downregulated TβRII and subsequent TGF-β signaling. Constitutively active MEK1, but not constitutively active ERK2, induced upregulation of TβRII. Furthermore, TβRII physically interacted with the constitutively active MEK1, but not with wild-type MEK1, indicating involvement of active MEK1 in stabilizing TβRII. Collectively, our data suggest a novel mechanism for MEK1 in regulating the sensitivity to TGF-β signaling by stabilizing TβRII.     

Expression and function of gingival fibroblast C1q receptors are upregulated by interleukin-1β and transforming growth factor-β

In injury and inflammation, complement (C) component C1q, in addition to its central role in initiation of classical pathway of complement activation, modulates diverse cellular functions by binding to specific cell surface receptors. Interaction of substrate-bound C1q with receptors for the collagen-like domain of C1q (C1qRC) of human gingival fibroblasts (HGF) promotes cell attachment. We investigated modulation of the adhesive function and expression of C1qRC by interleukin-1β (IL-1β) and transforming growth factor-β (TGF-β). Confluent fibroblast monolayers were incubated under standard culture conditions with or without cytokines. C1qRC function was measured by attachment assays. IL-1β and TGF-β increased fibroblast adhesion to C1q to 146% and 131% of controls, respectively. Cytokine enhancement of HGF adhesion was concentration-dependent, saturable (20 ng/ml IL-1β; 1 ng/ml TGF-β) and time-dependent (IL-1β 12-hr peak; TGF-β 24-hr peak). Effect of IL-1β and TGF-β on C1qRC expression was assessed by flow cytometry measurements of fluorescence intensity of cells stained with C1q and FITC anti-C1q antibody, and by binding studies with ¹²⁵l-C1q. Cells treated with cytokines displayed a two- to four-fold increased fluorescence of cell-bound C1q compared to controls. Binding studies indicated the increased fluorescence correlated with increase in number of C1qRC in both IL-1β (4.7 × 10⁶/cell) and TGF-β (3.9 × 10⁶/cell)-treated cells, compared to control (3.0 × 10⁶/cell), but had no effect on binding affinity. Rates of internalization of receptor-bound C1q were similar in cytokine-treated cells and controls. We propose from these data that IL-1β and TGF-β have the ability to upregulate C1qRC expression, and this effect contributes to increased adhesion of HGF to substrate-bound C1q. © 1993 Wiley-Liss, Inc.