TGF-βRII Expression Research Articles

Expression of individual members of the TGF-β/SMAD signalling pathway in the progression and survival of patients with colorectal carcinoma

Current knowledge of tumor biology offers many “targets” for therapeutic intervention. The molecular basis of many processes that play a role in the pathogenesis of colorectal cancer has been identified. One part of colorectal cancer clinical trials is focused on testing substances in a group of patients with tumors in which the TGF-β signalling pathway is hyperactivated. The TGF-β/SMAD signalling pathway members are considered important markers; however, genetic, proteomic, or metabolomic analyses still yield controversial results. According to our results, TGF-βRII, and SMAD4 can be used in monitoring CRC progression. With increasing CRC stage, TGF-βRII expression decreases and SMAD4 expression increases. The patients with TGF-βRII expression lower than 700 pg/ml had a slightly lower survival time (28.103 months) than patients with higher TGF-βRII expression (31.620 months). Conversely, patients with SMAD4 expression lower than 200 pg/ml had a higher survival rate (30.979 months) than patients with higher expression (26.316 months). Regarding TGF-β1 expression, the patient´s survival assessment determined no significant difference between patients with high or low tissue TGF-β1 expression. A personalized approach and consideration of a wide range of factors are important when using these markers in treatment assessment.

PD-L1 Acts Independently of PD-1 as a Marker of Pathologic Fibroblasts in Laryngotracheal Stenosis.

Laryngotracheal stenosis (LTS) describes fibrotic airway obstruction that is life-threatening without treatment. Targeted therapies are needed as an adjunct to surgical management. We have previously observed the upregulation of immune checkpoint programmed cell death (PD)-1 and its ligand, PD-L1, in patients with LTS. This study aims to determine whether PD-1 and PD-L1 play a role in the pathophysiology of LTS. Basic science. Laboratory. Fibroblasts derived from the subglottic scar of 5 iSGS patients were cultured ex vivo with transforming growth factor β (TGFβ), PD-L1 agonist (PD-1), and PD-L1 blockade (anti-PD-L1). PD-L1, TGFβ receptor II (TGFβRII), and Collagen-1 expression were quantified by flow cytometry. A validated chemomechanical injury model of subglottic stenosis was applied in PD-1 knockout and wild-type (WT) mice, and subglottic thickening was assessed by histologic analysis. TGFβ significantly increased the expression of PD-L1 and Collagen-1 in human airway scar fibroblasts (P < .05). PD-1 knockout mice demonstrated no significant difference in subglottic airway fibrosis compared to WT mice. Ex vivo PD-L1 modulation had no impact on fibroblast Collagen-1 expression. PD-L1 high-intensity fibroblasts expressed greater Collagen-1 and TGFβRII compared to PD-L1 low-intensity fibroblasts. PD-1 knockout does not protect mice from the development of laryngotracheal fibrosis. However, its ligand, PD-L1 is highly expressed on pathologic fibroblasts unique to scar, characterized by high Collagen-1 and TGFβRII expression. PD-L1 is also upregulated in conjunction with Collagen-1 by TGFβ stimulation. PD-L1 may act independently of PD-1 to sensitize fibroblasts to TGFβ, suggesting direct targeting of PD-L1 may have therapeutic potential in LTS.

Core needle biopsy changes the expression of TGFβ1 and TGFβRII at protein level, and the distribution of CD4 and CD8 positive T cells in primary breast cancer

Core needle biopsy (CNB) has become a paradigm in preoperative breast cancer (BC) diagnosis. Although considered safe, it is an invasive procedure, which changes the tumor microenvironment. It facilitates a tumor supportive immune response, induces epithelial-mesenchymal transition (EMT), and enables the release of circulating tumor cells. The cytokine Transforming Growth Factor β (TGFβ) with its pleiotropic immunologic functions has an important role in this process. The aim of this study was to clarify the specific impact of CNB on the activity of the TGFβ pathway in early BC. We compared formalin fixed paraffin embedded samples from CNBs to the corresponding surgical resection specimens (SRSs) of 49 patients with BC. We found that the expression of TGFβ1 at protein level was significantly higher in both tumor epithelial and benign stromal cells in the SRSs (p=0.001), whereas the expression of TGFβRII in tumor cells was lower (p=0.001). The frequency of intra tumoral CD8 and CD4 positive T lymphocytes was lower in SRSs (p=0081 and p=0001, respectively), while in the peripheral stroma their prevalence was increased (p=0001 and p=0012, respectively). Our results show that CNB changes the hallmarks of the TGFβ path way in early BC. These CNB-induced changes in the tumor and in its microenvironment suggest that the procedure may change the immunological anti-tumor response of the host.

Celastrol alleviates subconjunctival fibrosis induced by silicone implants mimicking glaucoma surgery

Subconjunctival fibrosis is critical to the outcomes of several ophthalmic conditions or procedures, such as glaucoma filtering surgery. This study aimed to investigate the anti-fibrotic effect of celastrol on subconjunctival fibrosis and to further reveal the underlying mechanisms. We used celastrol-loaded nanomicelles hydrogel hybrid as a sustained-release drug. A rabbit model of subconjunctival fibrosis following silicone implantation was used for in vivo study, and TGF-β1-induced human pterygium fibroblast (HPF) activation as an in vitro model. The effects of celastrol on inhibiting TGF-β1-induced migration and proliferation of HPFs were evaluated by scratch wound assay and CCK-8, respectively. Immunofluorescence and western blotting were used to examine the effect of celastrol on the expression of α-SMA, collagen I, fibronectin, and the targets of the Hippo signaling pathway. We found that in vivo celastrol treatment reduced the expression of YAP and TAZ in subconjunctival tissue. Moreover, celastrol alleviated collagen deposition and subconjunctival fibrosis at 8 weeks. No obvious tissue toxicity was observed in the rabbit models. Mechanistically, celastrol significantly inhibited TGF-β1-induced proliferation and migration of HPFs. Pretreatment of HPFs with celastrol also suppressed the TGF-β1-induced protein expression of α-SMA, collagen I, fibronectin, TGF-βRII, phosphorylated Smad2/3, YAP, TAZ, and TEAD1. In conclusion, celastrol effectively prevented subconjunctival fibrosis through inhibiting TGF-β1/Smad2/3-YAP/TAZ pathway. Celastrol could serve as a promising therapy for subconjunctival fibrosis.

Knockout of TGF-β receptor II by CRISPR/Cas9 delays mesenchymal transition of Lens epithelium and posterior capsule opacification

Posterior capsule opacification (PCO) is the most common postoperative complication of cataract surgery. Transforming growth factor-β (TGF-β) is related to epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) that is proven to induce PCO formation in clinical and experimental studies. In this study, CRISPR sequences targeting exon of TGF-βRII were knocked out with lentiviral transfection in LECs. Rabbits' PCO model was established and recombinant adeno-associated virus (AAV) for transferring the gRNA of TGF βRII were intravitreally injected. SgRNA inhibited TGF-βRII expression and human LECs proliferation. In TGF-βRII knockout group, LECs motility and migration were suppressed, N-cadherin and vimentin expressions were significantly decreased, whereas E-cadherin was increased. The animal model showed that TGF-βRII knockout in vivo was effective in suppressing PCO. The current study suggested that the CRISPR/Cas9 endonuclease system could suppress TGF-βRII secretion, which participates in the EMT procedure of LECs in vitro and PCO in vivo. These findings might provide a new gene-editing approach and insight into a novel therapeutic strategy for PCO.

CD47 Expression in Circulating Tumor Cells and Circulating Tumor Microemboli from Non-Small Cell Lung Cancer Patients Is a Poor Prognosis Factor.

To isolate, quantify and analyze CTCs/CTMs from metastatic NSCLC patients, identify TGFβRI/CD47 expression in CTCs/CTMs, and correlate with progression-free survival (PFS). Blood (10 mL) was collected at two time-points: T1 (before the beginning of any line of treatment; T2 (60 days after initial collection). CTCs were isolated using ISET®. Immunocytochemistry was conducted to evaluate TGFβRI/CD47 expression. 45 patients were evaluated. CTCs were observed in 82.2% of patients at T1 (median: 1 CTC/mL; range: 0.33-11.33 CTCs/mL) and 94.5% at T2 (median: 1.33 CTC/mL; 0.33-9.67). CTMs were observed in 24.5% of patients and significantly associated with poor PFS (10 months vs. 17 months for those without clusters; p = 0.05) and disease progression (p = 0.017). CTMs CD47+ resulted in poor PFS (p = 0.041). TGFβRI expression in CTCs/CTMs was not associated with PFS. In this study, we observed that CTC/CTM from NSCLC patients express the immune evasion markers TGFβRI/CD47. The presence of CTMs CD47+ is associated with poor PFS. This was the first study to investigate CD47 expression in CTCs/CTM of patients with NSCLC and its association with poor PFS.

Evaluation of the interaction between tumor growth factor-β and interferon type I pathways in patients with COVID-19: focusing on ages 1 to 90 years

BackgroundEvidence revealed that age could affect immune responses in patients with the acute respiratory syndrome of coronavirus 2 (SARS-CoV-2) infection. This study investigated the impact of age on immune responses, especially on the interaction between the tumor growth factor-β (TGF-β) and interferon type-I (IFN-I) axes in the pathogenesis of novel coronavirus disease 2019 (COVID-19).MethodsThis age-matched case–control investigation enrolled 41 COVID-19 patients and 40 healthy controls categorized into four groups, including group 1 (up to 20 years), group 2 (20–40 years), group 3 (40–60 years), and group 4 (over 60 years). Blood samples were collected at the time of admission. The expression of TGF-βRI, TGF-βRII, IFNARI, IFNARII, interferon regulatory factor 9 (IRF9), and SMAD family member 3 (SMAD3) was measured using the real-time PCR technique. In addition, serum levels of TGF-β, IFN-α, and SERPINE1 were measured by the enzyme-linked immunosorbent assay (ELISA) technique. All biomarkers were measured and analyzed in the four age studies groups.ResultsThe expression of TGF-βRI, TGF-βRII, IFNARI, IFNARII, IRF9, and SMAD3 was markedly upregulated in all age groups of patients compared with the matched control groups. Serum levels of IFN-α and SERPINE1 were significantly higher in patient groups than in control groups. While TGF-β serum levels were only significantly elevated in the 20 to 40 and over 60 years patient group than in matched control groups.ConclusionsThese data showed that the age of patients, at least at the time of admission, may not significantly affect TGF-β- and IFN-I-associated immune responses. However, it is possible that the severity of the disease affects these pathway-mediated responses, and more studies with a larger sample size are needed to verify it.

Celastrol Alleviates Corneal Stromal Fibrosis by Inhibiting TGF-β1/Smad2/3-YAP/TAZ Signaling After Descemet Stripping Endothelial Keratoplasty.

The purpose of this study was to investigate the effect of celastrol (CEL) on corneal stromal fibrosis after Descemet stripping endothelial keratoplasty (DSEK) and its associated mechanism. Rabbit corneal fibroblasts (RCFs) were isolated, cultured, and identified. A CEL-loaded positive nanomedicine (CPNM) was developed to enhance corneal penetration. CCK-8 and scratch assays were performed to evaluate cytotoxicity and the effects of CEL on the migration of RCFs. The RCFs were activated by TGF-β1 with or without CEL treatment, and then the protein expression levels of TGFβRII, Smad2/3, YAP, TAZ, TEAD1, α-SMA, TGF-β1, FN, and COLI were assessed by immunofluorescence or Western blotting (WB). An in vivo DSEK model was established in New Zealand White rabbits. The corneas were stained using H&E, YAP, TAZ, TGF-β1, Smad2/3, TGFβRII, Masson, and COLI. H&E staining of the eyeball was performed to assess the tissue toxicity of CEL at 8 weeks after DSEK. In vitro CEL treatment inhibited the proliferation and migration of RCFs induced by TGF-β1. Immunofluorescence and WB showed that CEL significantly inhibited the protein expression of TGF-β1, Smad2/3, YAP, TAZ, TEAD1, α-SMA, TGF-βRII, FN, and COL1 induced by TGF-β1 in RCFs. In the rabbit DSEK model, CEL significantly reduced the levels of YAP, TAZ, TGF-β1, Smad2/3, TGFβRII, and collagen. No obvious tissue toxicity was observed in the CPNM group. CEL effectively inhibited corneal stromal fibrosis after DSEK. The TGF-β1/Smad2/3-YAP/TAZ pathway may be involved in the mechanism by which CEL alleviates corneal fibrosis. The CPNM is a safe and effective treatment strategy for corneal stromal fibrosis after DSEK.

Molecular characterization and expression of TGFβRI and TGFβRII and its association with litter size in Tibetan sheep.

Transforming growth factor-β (TGF-β) type I receptor (TGFβRI) and type II receptor (TGFβRII) are the members of the TGFβ superfamily, which are potent regulators of cell proliferation and differentiation in many organ systems, and they play key roles in multiple aspects of follicle development. We aimed to explore the characterization, expression analysis of TGFβRI and TGFβRII genes, and the association with litter size in Tibetan sheep. In this study, we cloned the complete coding sequences of TGFβRI and TGFβRII genes in Tibetan sheep and analyzed their genomic structures. The results showed that percentages of sequences homology of the two proteins in Tibetan sheep were the most similar to Ovis aries (100%), followed by Bos mutus (99%). The RT-qPCR showed that two genes were expressed widely in the different tissues of Tibetan sheep. The TGFβRI expression was the highest in the lung (p < 0.05), followed by the spleen and ovary (p < 0.05). The TGFβRII expression was significantly higher in uterus than that in lung and ovary (p < 0.05). In addition, the χ2 test indicated that all ewes in the population were in Hardy-Weinberg equilibrium, and the population was in medium or low polymorphic information content status. We also found four Single Nucleotide Polymorphism (SNPs), g.9414A > G, g.28881A > G, g.28809T > C, g.10429G > A in sheep TGFβRI gene and g.63940C > T, g.63976C > T, g.64538C > T, g.64504T > A in TGFβRII gene. Three genotypes, except for g.64504T > A, and three haplotypes were identified in each gene. linkage disequilibrium analysis indicated that there was strong linkage disequilibrium in each gene. The association analysis showed that the four SNPs of TGFβRI were associated with litter size (p < 0.05), and g.63940C > T of TGFβRII was confirmed to be associated with litter size (p < 0.05). Based on these preliminary results, we can assume that TGFβ receptors (TGFβRI and TGFβRII) may play an important role in sheep reproduction.

Platycodin D inhibits the proliferation and migration of hypertrophic scar-derived fibroblasts and promotes apoptosis through a caspase-dependent pathway

Abnormal fibroblast proliferation and excessive extracellular matrix (ECM) deposition lead to the formation of hypertrophic scars (HSs). However, there is no satisfactory method to inhibit the occurrence and development of HSs. In our study, platycodin D (PD), a natural compound extracted from Platycodon grandiflorus, inhibited HSs formation both in vitro and in vivo. First, qRT-PCR and Western blot were used to confirm PD dose-dependently downregulated the expression of Col I, Col III and α-SMA in human hypertrophic scar-derived fibroblasts (HSFs) (p < 0.05). Second, cck-8, transwell and wound healing assays verified PD suppressed the proliferation (p < 0.05) and migration of HSFs (p < 0.05), and inhibited the differentiation of HSFs into myofibroblasts. Moreover, PD-induced HSFs apoptosis were analyzed by flow cytometry and the apoptosis was activated through a caspase-dependent pathway. The rabbit ear scar model was used to further confirm the inhibitory effect of PD on collagen and α-SMA deposition. Finally, Western blot analysis showed that PD reduced TGF-β RI expression (p < 0.05) and affected matrix metalloproteinase 2 (MMP2) protein levels (p < 0.05). In conclusion, our study showed that PD inhibited the proliferation and migration of HSFs by inhibiting fibrosis-related molecules and promoting apoptosis via a caspase-dependent pathway. The TGF-β/Smad pathway also mediated the inhibition of HSFs proliferation and HSFs differentiation into myofibroblasts. Therefore, PD is a potential therapeutic agent for HSs and other fibrotic diseases.

Numbers of Follicles, Pregnancy, Expression of Reactive Oxygen Species (ROS), and Ovarian Genes in Sheep Treated with Vitamin E, L-Carnitine, and Fish Oil

This study was performed to evaluate the effects of vitamin E, L-carnitine, and fish oil on the numbers of follicles, pregnancy, Reactive Oxygen Species (ROS) expression, and expression of several ovarian genes in sheep. For this purpose, 256 sheep were randomly divided into eight experimental groups. They were given a diet supplemented with vitamin E, L-carnitine, and fish oil alone and in combination. FBS, estradiol levels, size of the follicles, and pregnancy rate were observed. Expressions of Superoxide Dismutase 1 ( SOD1 ), Superoxide Dismutase 2 ( SOD2 ), Transforming Growth Factor –β ( TGF-β ), and Peroxisome Proliferator-Activated Gamma Receptor ( PPAR-γ ) genes were measured using Reverse Transcription-Polymerase Chain Reaction (RT-PCR), and ROS was measured using fluorescence microscope. At the time of mating and pregnancy, the highest number of follicles and pregnant ewes were observed in the groups supplemented with fish oil and antioxidants (p<0.01). The lowest numbers of follicles and pregnancy was observed in the group supplemented with fish oil alone (p<0.01). The highest expressions of SOD1 and TGF-β genes (p<0.01) as well as SOD2 and PPAR-γ (p<0.05) were observed in the group of concomitant use of fish oil with antioxidants. The highest amount of ROS was found in fish oil group (p<0.01), and the lowest was found in the groups supplemented with fish oil and antioxidant (p<0.01). The use of fish oil along with vitamin E and L-carnitine improved follicle function and increased pregnancy rate by reducing ROS in ewes’ ovaries as well as increasing the expression of SOD1 , SOD2 , TGF-βRI , and PPAR-γ genes. The use of fish oil along with antioxidants increases follicles and improves fertility in sheep.

Suppression of CD56bright NK cells in breast cancer patients is associated with the PD-1 and TGF-βRII expression.

Natural killer (NK) cells, as professional cytotoxic cells, play a key role againstcancer intheearly and metastaticstages. Their functional defects are highly associated with the initiation or progression of breast cancer (BC). Here, we investigated the phenotypic characterization of NK cells in 26 newly diagnosed BC patients in comparison to 12 healthy counterparts. Expression of CXCR3 and PD-1, and also NKG2D, and TGF-βRII were studied on CD56dim and CD56bright NK cells from fresh peripheral blood (PB) samples using flow cytometry. The plasma levels of IFN-γ and soluble MIC-A levels were also assessed by ELISA. Both CD56dim and CD56bright NK subtypes showed lower CXCR3 and NKG2D expression in BC patients than healthy subjects. Furthermore, patients' CD56bright NK cells significantly showed higher expression levels of TGF-βRII and PD-1. Interestingly, increased concentration of MIC-A level in plasma of BC patients was associated with the higher TGF-βRII and PD-1 expression in all NK cells, while the plasma level of IFN-γ was associated with the lower TGF-βRII expression on CD56bright NK cells in these patients. Our results demonstrated phenotypically suppressed-NK cells, especially in the CD56bright subset of BC patients. It specifies their potential incompetence and outlines decrement of their anti-tumor activity, which could be interrelated with the tumor pathogenesis, TME immunosuppression, and so disease progression. The induction of compensatory mechanisms revives NK cells function and could be used in combination with the conventional treatments as a putative therapeutic approach for targeted immunotherapy.

Altered expression of activating transcription factor 3 in the hippocampus of patients with mesial temporal lobe epilepsy-hippocampal sclerosis (MTLE-HS)

Aim of the study: Activating Transforming factor 3 (ATF3) is a stress induced gene and closely associated with neuro-inflammation while Transforming growth Factor Beta (TGFβ) signalling is also reported to be involved in neuro-inflammation and hyper-excitability associated with drug resistant epilepsy. Animal model studies indicate the involvement of ATF3 and TGFβ receptors to promote epileptogenesis. Human studies also show that TGFβ signalling is activated in MTLE-HS. However, lack of studies on ATF3 and TGFβRI expression in MTLE-HS patients exists. We hypothesize that ATF3 and TGFβRI might be expressed in hippocampi of patients with MTLE-HS and playing role in epileptogenesis. Materials & methods: Protein expression of ATF3 and TGFβRI was performed by western blotting. Localisation of ATF3 was performed by immunohistochemistry and immunoflorescence. Results: Protein expression of ATF3 and TGFβRI was significantly up-regulated in hippocampi of patients as compared to controls. Also ATF3 IR was significantly expressed in hippocampi of patients and ATF3 was expressed predominantly in cytoplasm as compared to nucleus. No correlation was found between ATF3 expression and epilepsy duration and seizure frequency. Conclusions: ATF3 and TGFβRI are both important players in neuro-inflammation and might potentiate epileptogenesis in these patients.

Ochratoxin a induces hepatic fibrosis through TGF-β receptor I/Smad2/3 signaling pathway.

Ochratoxin A (OTA) is a mycotoxin generated by Penicillium and Aspergillus species. It is often found in cereals. We hypothesized that OTA exposure induces epithelial-mesenchymal transition (EMT), leading to liver fibrosis. In this research, we explored whether the TGF-β receptor I (TGF-β RI)/Smad2/3 signaling pathway is related to EMT-induced hepatic fibrosis. In vitro and in vivo experiments, mRNA and protein expression of liver fibrosis-related markers such as fibronectin, α-smooth muscle actin (α-SMA) and E-cadherin were assessed. The levels of alkaline phosphatase, alanine transaminase, aspartate aminotransferase, and total bilirubin, which are used to assess damage, increased. We also confirmed the increase in mRNA and protein expression of TGF-β RI, Smad2, and Smad3. The expression of liver fibrosis-related markers was decreased by siRNA-mediated silencing of Smad2/3, as well as TGF-RI suppression. Liver cells exposed to OTA showed enhanced TGF-β RI expression on the cell membrane. These results demonstrated that OTA induces hepatic fibrosis through TGF-β RI and Smad2/3 pathways in vitro and in vivo.

Endothelial Microparticle-Mediated Transfer of microRNA-19b Inhibits the Function and Distribution of Lymphatic Vessels in Atherosclerotic Mice

In recent years, the function of the lymphatic system in atherosclerosis has attracted attention due to its role in immune cell trafficking, cholesterol removal from the periphery, and regulation of the inflammatory response. However, knowledge of the mechanisms regulating lymphangiogenesis and lymphatic function in the pathogenesis of atherosclerosis is limited. Endothelial microparticles carrying circulating microRNA (miRNA)s are known to mediate cell–cell communication, and our previous research showed that miRNA-19b in EMPs (EMPmiR-19b) was significantly increased in circulation and atherosclerotic vessels, and this increase in EMPmiR-19b promoted atherosclerosis. The present study investigated whether atherogenic EMPmiR-19b influences pathological changes of the lymphatic system in atherosclerosis. We first verified increased miR-19b levels and loss of lymphatic system function in atherosclerotic mice. Atherogenic western diet-fed ApoE-/- mice were injected with phosphate-buffered saline, EMPs carrying control miRNA (EMPcontrol), or EMPmiR-19b intravenously. The function and distribution of the lymphatic system was assessed via confocal microscopy, Evans blue staining, and pathological analysis. The results showed that lymphatic system dysfunction existed in the early stage of atherosclerosis, and the observed pathological changes persisted at the later stage, companied by an increased microRNA-19b level. In ApoE-/- mice systemically treated with EMPmiR-19b, the distribution, transport function, and permeability of the lymphatic system were significantly inhibited. In vitro experiments showed that miRNA-19b may damage the lymphatic system by inhibiting lymphatic endothelial cell migration and tube formation, and a possible mechanism is the inhibition of transforming growth factor beta receptor type II (TGF-βRII) expression in lymphatic endothelial cells by miRNA-19b. Together, our findings demonstrate that atherogenic EMPmiR-19b may destroy lymphatic system function in atherosclerotic mice by downregulating TGF-βRII expression.

Gp130-Mediated STAT3 Activation Contributes to the Aggressiveness of Pancreatic Cancer through H19 Long Non-Coding RNA Expression.

Simple SummaryThe signal transducer and activator of transcription 3 (STAT3) activation correlate with the aggressiveness of pancreatic ductal adenocarcinoma (PDAC). We demonstrated that the autocrine/paracrine interleukin-6 (IL-6) or leukemia inhibitory factor (LIF)/glycoprotein 130 (gp130)/STAT3 pathway contributes to the maintenance of stemness features and membrane-type 1 matrix metalloproteinase (MT1-MMP) expression, and modulates transforming growth factor (TGF)-β1/Smad signaling-mediated epithelial-mesenchymal transition (EMT) and invasion through regulation of TGFβ-RII expression in PDAC cancer stem cell (CSC)-like cells. Furthermore, we demonstrated that p-STAT3 acts through the IL-6 or LIF/gp130/STAT3 pathway to access the active promoter region of metastasis-related long non-coding RNA H19 and contribute to its transcription in CSC-like cells. Therefore, the autocrine/paracrine IL-6 or LIF/gp130/STAT3 pathway in PDAC CSC-like cells exhibiting H19 expression is considered to be involved in the aggressiveness of PDAC, and inhibition of the gp130/STAT3 pathway is a promising strategy to target CSCs for the elimination of PDAC (146/150).Signaling pathways involving signal transducer and activator of transcription 3 (STAT3) play key roles in the aggressiveness of pancreatic ductal adenocarcinoma (PDAC), including their tumorigenesis, invasion, and metastasis. Cancer stem cells (CSCs) have been correlated with PDAC aggressiveness, and activation of STAT3 is involved in the regulation of CSC properties. Here, we investigated the involvement of interleukin-6 (IL-6) or the leukemia inhibitory factor (LIF)/glycoprotein 130 (gp130)/STAT3 pathway and their role in pancreatic CSCs. In PDAC CSC-like cells formed by culturing on a low attachment plate, autocrine/paracrine IL-6 or LIF contributes to gp130/STAT3 pathway activation. Using a gp130 inhibitor, we determined that the gp130/STAT3 pathway contributes to the maintenance of stemness features, the expression of membrane-type 1 matrix metalloproteinase (MT1-MMP), and the invasion of PDAC CSC-like cells. The gp130/STAT3 pathway also modulates the transforming growth factor (TGF)-β1/Smad pathway required for epithelial-mesenchymal transition induction through regulation of TGFβ-RII expression in PDAC CSC-like cells. Furthermore, chromatin immunoprecipitation assays revealed that p-STAT3 can access the active promoter region of H19 to influence this metastasis-related long non-coding RNA and contribute to its transcription in PDAC CSC-like cells. Therefore, the autocrine/paracrine IL-6 or LIF/gp130/STAT3 pathway in PDAC CSC-like cells may eventually facilitate invasion and metastasis, two hallmarks of malignancy. We propose that inhibition of the gp130/STAT3 pathway provides a promising strategy for targeting CSCs for the treatment of PDAC.

Evaluation of effective factors on IL-10 signaling in B cells in patients with selective IgA deficiency

Selective IgA deficiency is the most prevalent form of primary immunodeficiencies. The pathogenesis of the disease is still unknown. Several studies have suggested a defect in B cell responses to IL-10; however, the main reason for this defect has not been reported. Elucidating IL-10 signaling defects and their correlation with clinical manifestations could be helpful for better understanding and treatment of the disease. In this study, 15 SIgAD patients and 15 age- and sex-matched healthy controls were included. Surface expression of transforming growth factor β receptor II (TGF-β RII), IL-10R and IgA was assessed by flow cytometry in human purified B cells before and after stimulation by IL-10. Protein expression of STAT3, p-STAT3 and SOCS3 was measured by Western blotting analysis. TGF-β and IgA secretion was evaluated by ELISA. Finally, the measurement of B cell apoptosis was performed by flow cytometry. The TGF-βRII expression level was decreased after stimulation with IL-10 in patients compared with controls. Notably, the TGF-β level were higher after stimulation with mCD40L and IL-10 in the control group as compared to stimulation with mCD40L alone. The IgA+ B cell percentage and IgA secretion levels were significantly increased in controls as compared with SIgAD patients. The relative concentration of the total STAT3 was decreased as compared with controls. The defect in IgA production in SIgAD patients could be due to inadequate B cell responses to IL-10 stimulation that probably originate from defective regulation of IL-10-mediated TGF-b ’symbol’ production TGF-β response by IL-10. Furthermore, it is suggested that the absence of STAT3 protein baseline expression could impair cytokine-mediated signaling such as thatinduced by IL-!0 and IL-21.

Estradiol Ameliorates Acute Kidney Ischemia-Reperfusion Injury by Inhibiting the TGF-βRI-SMAD Pathway.

Renal ischemia–reperfusion injury (IRI) is less extensive in females than males in both animals and humans; however, this protection diminishes after menopause, suggesting that estrogen plays a pivotal role in IRI, but the underlying mechanism remains largely unknown. Our study found that 45 min of warm ischemia was sufficient to induce significant pathological changes without causing death in model animals. Compared with male rats, female rats exhibited less extensive apoptosis, kidney injury, and fibrosis; these effects were worsened in ovariectomized (OVX) rats and ameliorated upon estradiol (E2) supplementation. Furthermore, the levels of TGF-βRI, but not TGF-βRII or TGF-β1, were significantly increased in OVX rats, accompanied by phosphorylated SMAD2/3 activation. Interestingly, the alteration trend of the nuclear ERα level was opposite that of TGF-βRI. Furthermore, dual luciferase reporter and chromatin immunoprecipitation assays showed that ERα could bind to the promoter region of TGF-βRI and negatively regulate its mRNA expression. Moreover, an in vitro study using NRK-52E cells showed that ERα knockdown blocked E2-mediated protection, while TGF-βRI knockdown protected cells against hypoxic insult. The findings of this study suggest that renal IRI is closely related to the TGF-βRI-SMAD pathway in females and that E2 exert its protective effect via the ERα-mediated transcriptional inhibition of TGF-βRI expression.

Prognostic Relevance of Transforming Growth Factor-β Receptor Expression and Signaling in Glioblastoma, Isocitrate Dehydrogenase-Wildtype.

The transforming growth factor (TGF)-β signaling pathway has been recognized as a major factor in promoting the aggressive behavior of glioblastoma, isocitrate dehydrogenase-wildtype. However, there is little knowledge about the expression of TGF-β receptors in glioblastoma. Here, we studied the expression patterns of TGF-β receptor II (TGFβRII), type I receptors activin receptor-like kinase (ALK)-5, and ALK-1, as well as of the transcriptional regulators inhibitor of differentiation (Id) 2, Id3, and Id4 in human glioblastoma. The expression of TGFβRII, ALK-5, and ALK-1 varied greatly, with TGFβRII and ALK-5 being the most abundant and ALK-1 being the least expressed receptor. None of the 3 receptors was preferentially expressed by tumor vasculature as opposed to the tumor bulk, indicating tumor bulk-governed mechanisms of TGF-β signaling with regard to glioblastoma-associated angiogenesis. A positive correlation was found between ALK-1 and Id2, suggesting that Id2, broadly expressed in the tumor cells, is a downstream target of this receptor-dependent pathway. Furthermore, there was a trend for high expression of ALK-5 or Id2 to be associated with inferior overall survival. Hence, we propose that ALK-5 may be used for patient stratification in future anti-TGF-β treatment trials and that Id2 might be a potential target for anti-TGF-β interventions.

Smurf participates in Helicoverpa armigera diapause by regulating the transforming growth factor-β signaling pathway.

Diapause, an important strategy used by insects to avoid adverse environments, is regulated by various cell signaling pathways. The results of our previous studies demonstrated that the transforming growth factor-β (TGF-β) signaling pathway regulated pupal diapause in Helicoverpa armigera, which was accompanied by downregulation of proteins in TGF-β signaling. However, to date the mechanism underlying this phenomenon remains unknown. Here, we cloned the E3 ubiquitin ligases gene Smurf. In vitro experiments showed that Smurf directly bound to TGF-β receptor type I (TGFβRI) and Smad2. Overexpressing Smurf promoted ubiquitination of TGFβRI and Smad2, thereby downregulating their protein levels. Conversely, silencing of the Smurf gene suppressed ubiquitination of TGFβRI and Smad2 thereby increasing their protein levels. Results from in vivo co-immunoprecipitation assays revealed that the binding of Smurf to TGFβRI or Smad2 was stronger in diapause pupae than in nondiapause pupae. Injection of Smurf inhibitor A01 into diapause pupae markedly upregulated expression of TGFβRI and Smad2 proteins, leading to resumption of development in diapause pupae. Taken together, these findings suggested that ubiquitin ligase E3 Smurf participated in H. armigera diapause by regulating TGF-β signaling, and thus could be playing a crucial role in insect diapause.