PARP-1 regulates expression of TGF-? receptors in T cells.
ABSTRACT: Transforming growth factor-? (TGF-?) receptors (T?Rs) are essential components for TGF-? signal transduction in T cells, yet the mechanisms by which the receptors are regulated remain poorly understood. We show here that Poly(ADP-ribose) polymerase-1 (PARP-1) regulates TGF-? receptor I (T?RI) and II (T?RII) expression in CD4(+) T cells and subsequently affects Smad2/3-mediated TGF-? signal transduction. Inhibition of PARP-1 led to the upregulation of both T?RI and T?RII, yet the underlying molecular mechanisms were distinct. PARP-1 selectively bound to the promoter of T?RII, whereas the enzymatic activity of PARP-1 was responsible for the inhibition of T?RI expression. Importantly, inhibition of PARP-1 also enhanced expression of T?Rs in human CD4(+) T cells. Thus, PARP-1 regulates T?R expression and TGF-? signaling in T cells.
Project description:Transforming growth factor-?s (TGF-?s) regulate cellular proliferation, differentiation, and survival. TGF-?s bind to type I (TGF-?RI) and II receptors (TGF-?RII), which are transmembrane kinase receptors, and an accessory type III receptor (TGF-?RIII). TGF-? may utilize another type I receptor, activin-like kinase receptor (Alk1). TGF-? is neuroprotective in the middle cerebral artery occlusion (MCAO) model of stroke. Recently, we reported the expression pattern of TGF-?1-3 after MCAO. To establish how TGF-?s exert their actions following MCAO, the present study describes the induction of TGF-?RI, RII, RIII and Alk1 at 24 h, 72 h and 1 mo after transient 1 h MCAO as well as following 24 h permanent MCAO using in situ hybridization histochemistry. In intact brain, only TGF-?RI had significant expression: neurons in cortical layer IV contained TGF-?RI. At 24 h after the occlusion, no TGF-? receptors showed induction. At 72 h following MCAO, all four types of TGF-? receptors were induced in the infarct area, while TGF-?RI and RII also appeared in the penumbra. Most cells with elevated TGF-?RI mRNA levels were microglia. TGF-?RII co-localized with both microglial and endothelial markers while TGF-?RIII and Alk1 were present predominantly in endothels. All four TGF-? receptors were induced within the lesion 1 mo after the occlusion. In particular, TGF-?RIII was further induced as compared to 72 h after MCAO. At this time point, TGF-?RIII signal was predominantly not associated with blood vessels suggesting its microglial location. These data suggest that TGF-? receptors are induced after MCAO in a timely and spatially regulated fashion. TGF-? receptor expression is preceded by increased TGF-? expression. TGF-?RI and RII are likely to be co-expressed in microglial cells while Alk1, TGF-?RII, and RIII in endothels within the infarct where TGF-?1 may be their ligand. At later time points, TGF-?RIII may also appear in glial cells to potentially affect signal transduction via TGF-?RI and RII.
Project description:Tumor progression is governed by various growth factors and cytokines in the tumor microenvironment (TME). Among these, transforming growth factor-? (TGF-?) is secreted by various cell types residing in the TME and promotes tumor progression by inducing the epithelial-to-mesenchymal transition (EMT) of cancer cells and tumor angiogenesis. TGF-? comprises three isoforms, TGF-?1, -?2, and -?3, and transduces intracellular signals via TGF-? type I receptor (T?RI) and TGF-? type II receptor (T?RII). For the purpose of designing ligand traps that reduce oncogenic signaling in the TME, chimeric proteins comprising the ligand-interacting ectodomains of receptors fused with the Fc portion of immunoglobulin are often used. For example, chimeric soluble T?RII (T?RII-Fc) has been developed as an effective therapeutic strategy for targeting TGF-? ligands, but several lines of evidence indicate that T?RII-Fc more effectively traps TGF-?1 and TGF-?3 than TGF-?2, whose expression is elevated in multiple cancer types. In the present study, we developed a chimeric TGF-? receptor containing both T?RI and T?RII (T?RI-T?RII-Fc) and found that T?RI-T?RII-Fc trapped all TGF-? isoforms, leading to inhibition of both the TGF-? signal and TGF-?-induced EMT of oral cancer cells, whereas T?RII-Fc failed to trap TGF-?2. Furthermore, we found that T?RI-T?RII-Fc suppresses tumor growth and angiogenesis more effectively than T?RII-Fc in a subcutaneous xenograft model of oral cancer cells with high TGF-? expression. These results suggest that T?RI-T?RII-Fc may be a promising tool for targeting all TGF-? isoforms in the TME.
Project description:This study focused on characterizing the potential mechanism of valvular toxicity caused by TGF? receptor inhibitors (TGF?Ris) using rat valvular interstitial cells (VICs) to evaluate early biological responses to TGF?R inhibition. Three TGF?Ris that achieved similar exposures in the rat were assessed. Two dual TGF?RI/-RII inhibitors caused valvulopathy, whereas a selective TGF?RI inhibitor did not, leading to a hypothesis that TGF? receptor selectivity may influence the potency of valvular toxicity. The dual valvular toxic inhibitors had the most profound effect on altering VIC phenotype including altered morphology, migration, and extracellular matrix production. Reduction of TGF? expression demonstrated that combined TGF?2/?3 inhibition by small interfering RNA or neutralizing antibodies caused similar alterations as TGF?Ris. Inhibition of <i>TGF?3</i> transcription was only associated with the dual TGF?Ris, suggesting that TGF?RII inhibition impacts <i>TGF?3</i> transcriptional regulation, and that the potency of valvular toxicity may relate to alteration of TGF?2/?3-mediated processes involved in maintaining proper balance of VIC phenotypes in the heart valve.
Project description:Background:TGF-? isoforms play crucial roles in diverse cellular processes. Therefore, targeting and inhibiting TGF-? signaling pathway provides a potential therapeutic opportunity. TGF-? isoforms bind and bring the receptors (T?RII and T?RI) together to form a signaling complex in an ordered manner. Objectives:Herein, an antagonistic variant of TGF-? (AnT?) has been designed and prepared to inhibit the formation of signaling complex and consequently its signaling pathway. This TGF-? homodimeric variant contains intact T?RII binding sites and blocked T?RI binding sites by substituting three peptide segments. So, AnT? could only bind to T?RII, but prevent binding and recruitment of T?RI to form a signaling complex. Materials and Methods:A reliable model of AnT? was built and re?ned using molecular dynamics (MD) simulation, followed by investigating the interactions of AnT? with the receptors using in silico docking studies. After expression of disulfide-linked AnT? in a SHuffle strain and purification of the protein using affinity chromatography, its biological activity was evaluated using Mink lung epithelial cells (Mvl Lu). Results:No meaningful significant changes in AnT? structure were observed when compared with the native protein. Based on the docking analysis, AnT? binds to T?RII similar to TGF-? and its binding to T?RI was diminished considerably which was consistent with our design purpose. Cell-based bioassay indicated that AnT? could modulate TGF-?-induced cell growth inhibition. Conclusions:Our analysis suggests that the antagonistic potency of AnT? can be used as an anti-TGF? signaling factor in the future perspectives.
Project description:<h4>Rationale</h4>Chronic thromboembolic pulmonary hypertension (CTEPH) is characterized by defective thrombus resolution, pulmonary artery obstruction, and vasculopathy. TGF? (transforming growth factor-?) signaling mutations have been implicated in pulmonary arterial hypertension, whereas the role of TGF? in the pathophysiology of CTEPH is unknown.<h4>Objective</h4>To determine whether defective TGF? signaling in endothelial cells contributes to thrombus nonresolution and fibrosis.<h4>Methods and results</h4>Venous thrombosis was induced by inferior vena cava ligation in mice with genetic deletion of TGF?1 in platelets (Plt.TGF?-KO) or TGF? type II receptors in endothelial cells (End.TGF?RII-KO). Pulmonary endarterectomy specimens from CTEPH patients were analyzed using immunohistochemistry. Primary human and mouse endothelial cells were studied using confocal microscopy, quantitative polymerase chain reaction, and Western blot. Absence of TGF?1 in platelets did not alter platelet number or function but was associated with faster venous thrombus resolution, whereas endothelial TGF?RII deletion resulted in larger, more fibrotic and higher vascularized venous thrombi. Increased circulating active TGF?1 levels, endothelial TGF?RI/ALK1 (activin receptor-like kinase), and TGF?RI/ALK5 expression were detected in End.TGF?RII-KO mice, and activated TGF? signaling was present in vessel-rich areas of CTEPH specimens. CTEPH-endothelial cells and murine endothelial cells lacking TGF?RII simultaneously expressed endothelial and mesenchymal markers and transcription factors regulating endothelial-to-mesenchymal transition, similar to TGF?1-stimulated endothelial cells. Mechanistically, increased endothelin-1 levels were detected in TGF?RII-KO endothelial cells, murine venous thrombi, or endarterectomy specimens and plasma of CTEPH patients, and endothelin-1 overexpression was prevented by inhibition of ALK5, and to a lesser extent of ALK1. ALK5 inhibition and endothelin receptor antagonization inhibited mesenchymal lineage conversion in TGF?1-exposed human and murine endothelial cells and improved venous thrombus resolution and pulmonary vaso-occlusions in End.TGF?RII-KO mice.<h4>Conclusions</h4>Endothelial TGF?1 signaling via type I receptors and endothelin-1 contribute to mesenchymal lineage transition and thrombofibrosis, which were prevented by blocking endothelin receptors. Our findings may have relevant implications for the prevention and management of CTEPH.
Project description:Transforming growth factor-beta (TGF-beta) signaling is disrupted in many cancers, including cervical cancer, leading to TGF-beta resistance. Although initially sensitive, human papillomavirus type 16 (HPV16) immortalized human keratinocytes (HKc/HPV16) become increasingly resistant to the growth inhibitory effects of TGF-beta during in vitro progression to a differentiation resistant phenotype (HKc/DR). We have previously shown that loss of TGF-beta sensitivity in HKc/DR is attributed to decreased expression of TGF-beta receptor type I (TGF-beta RI), while the levels of TGF-beta receptor type II (TGF-beta RII) remain unchanged. The present study explored molecular mechanisms leading to reduced TGF-beta RI expression in HKc/DR. Using TGF-beta RI and TGF-beta RII promoter reporter constructs, we determined that acute expression of the HPV16 oncogenes E6 and E7 decreased the promoter activity of TGF-beta RI and TGF-beta RII by about 50%. However, promoter activity of TGF-beta RI is decreased to a greater extent than TGF-beta RII as HKc/HPV16 progress to HKc/DR. Reduced TGF-beta RI expression in HKc/DR was found not to be linked to mutations within the TGF-beta RI promoter or to promoter methylation. Electrophoretic mobility shift and supershift assays using probes encompassing Sp1 binding sites in the TGF-beta RI promoter found no changes between HKc/HPV16 and HKc/DR in binding of the transcription factors Sp1 or Sp3 to the probes. Also, Western blots determined that protein levels of Sp1 and Sp3 remain relatively unchanged between HKc/HPV16 and HKc/DR. Overall, these results demonstrate that mutations in or hypermethylation of the TGF-beta RI promoter, along with altered levels of Sp1 or Sp3, are not responsible for the reduced expression of TGF-beta RI we observe in HKc/DR. Rather the HPV16 oncogenes E6 and E7 themselves exhibit an inhibitory effect on TGF-beta receptor promoter activity.
Project description:Our previous studies have shown that Peyer's patches (PPs) play a key role in the induction of oral tolerance. Therefore, we hypothesized that PPs are an important site for Transforming Growth Factor (TGF)-? signaling and sought to prove that this tissue is of importance in oral tolerance induction. We found that expression of TGF-? type II receptor (TGF?RII) by CD4(+) T cells increases and persists in the PPs of normal C57BL/6 mice after either high- or low-dose feeding of OVA when compared to mesenteric lymph nodes (MLNs) and spleen. Approximately one-third of these TGF?RII(+) CD4(+) T cells express the transcription factor Foxp3. Interestingly, the number of TGF?RII(+) CD4(+) T cells in PPs decreased when OVA-fed mice were orally challenged with OVA plus native cholera toxin (CT). In contrast, numbers of TGF?RII(+) CD4(+) T cells were increased in the intestinal lamina propria (iLP) of these challenged mice. Further, these PP CD4(+) TGF?RII(+) T cells upregulated Foxp3 within 2 hours after OVA plus CT challenge. Mice fed PBS and challenged with OVA plus CT did not reveal any changes in TGF?RII expression by CD4(+) T cells. In order to test the functional property of TGF?RII in the induction of oral tolerance, CD4dnTGF?RII transgenic mice, in which TGF?RII signaling is abrogated from all CD4(+) T cells, were employed. Importantly, these mice could not develop oral tolerance to OVA. Our studies show a critical, dose-independent, role for TGF?RII expression and function by CD4(+) T cells in the gut-associated lymphoid tissues, further underlining the vital role of PPs in oral tolerance.
Project description:Transforming growth factor (TGF)-?s are dimeric polypeptides that have vital roles in regulating cell growth and differentiation. They signal by assembling a receptor heterotetramer composed of two T?RI:T?RII heterodimers. To investigate whether the two heterodimers bind and signal autonomously, one of the TGF-? protomers was substituted to block receptor binding. The substituted dimer, TGF-?3 WD, bound the T?RII extracellular domain and recruited the T?RI with affinities indistinguishable from TGF-?3, but with one-half the stoichiometry. TGF-?3 WD was further shown to retain one-quarter to one-half the signalling activity of TGF-?3 in three established assays for TGF-? function. Single-molecule fluorescence imaging with GFP-tagged receptors demonstrated a measurable increase in the proportion of T?RI and T?RII dimers upon treatment with TGF-?3, but not with TGF-?3 WD. These results provide evidence that the two T?RI:T?RII heterodimers bind and signal in an autonomous manner. They further underscore how the TGF-?s diverged from the bone morphogenetic proteins, the ancestral ligands of the TGF-? superfamily that signal through a RI:RII:RII heterotrimer.
Project description:Transforming growth factor (TGF) ?1, ?2, and ?3 (TGF-?1-TGF-?3, respectively) are small secreted signaling proteins that each signal through the TGF-? type I and type II receptors (T?RI and T?RII, respectively). However, TGF-?2, which is well-known to bind T?RII several hundred-fold more weakly than TGF-?1 and TGF-?3, has an additional requirement for betaglycan, a membrane-anchored nonsignaling receptor. Betaglycan has two domains that bind TGF-?2 at independent sites, but how it binds TGF-?2 to potentiate T?RII binding and how the complex with TGF-?, T?RII, and betaglycan undergoes the transition to the signaling complex with TGF-?, T?RII, and T?RI are not understood. To investigate the mechanism, the binding of the TGF-?s to the betaglycan extracellular domain, as well as its two independent binding domains, either directly or in combination with the T?RI and T?RII ectodomains, was studied using surface plasmon resonance, isothermal titration calorimetry, and size-exclusion chromatography. These studies show that betaglycan binds TGF-? homodimers with a 1:1 stoichiometry in a manner that allows one molecule of T?RII to bind. These studies further show that betaglycan modestly potentiates the binding of T?RII and must be displaced to allow T?RI to bind. These findings suggest that betaglycan functions to bind and concentrate TGF-?2 on the cell surface and thus promote the binding of T?RII by both membrane-localization effects and allostery. These studies further suggest that the transition to the signaling complex is mediated by the recruitment of T?RI, which simultaneously displaces betaglycan and stabilizes the bound T?RII by direct receptor-receptor contact.
Project description:Targeting tumor microenvironment (TME) is crucial in order to overcome the anti-cancer therapy resistance. In this study, we report the antitumor activity of a newly synthesized ?-carboline derivative "B-9-3." Here, this small molecule showed a promising antitumor activity in vivo along with an enhanced immune response as reflected by a reduction of regulatory T cells and increased CD4+/CD8+ T cells. Further, B-9-3 decreased the number of myofibroblasts not only in the tumor but also in the lung suggesting an anti-metastatic action. The reduction of myofibroblasts was associated with lower expression of epithelial-to-mesenchymal transition markers and a decrease of phosphorylated SMAD2/3 complex indicating the implication of TGF-? signaling pathway in B-9-3's effect. The blockade of myofibroblasts induction by B-9-3 was also verified in vitro in human fibroblasts treated with TGF-?. To elucidate the mechanism of B-9-3's action on TGF-? pathway, first, we investigated the molecular interaction between B-9-3 and TGF-? receptors using docking method. Data showed a weak interaction of B-9-3 with the ATP-binding pocket of TGF?RI but a strong one with a ternary complex formed of extracellular domains of TGF?RI, TGF?RII, and TGF-?. In addition, the role of TGF?RI and TGF?RII in B-9-3's activity was explored in vitro. B-9-3 did not decrease any of the two receptors' protein level and only reduced phosphorylated SMAD2/3 suggesting that its effect was more probably due to its interaction with the ternary complex rather than decreasing the expression of TGF-? receptors or interfering with their ATP-binding domains. B-9-3 is a small active molecule which acts on the TGF-? signaling pathway and improves the TME to inhibit the proliferation and the metastasis of the tumor with the potential for clinical application.