Sensing of ATP via the Purinergic Receptor P2RX7 Promotes CD8+ Trm Cell Generation by Enhancing Their Sensitivity to the Cytokine TGF-ß.

Tissue-resident memory (Trm) CD8+ T cells mediate protective immunity in barrier tissues, but the cues promoting Trm cell generation are poorly understood. Sensing of extracellular adenosine triphosphate (eATP) by the purinergic receptor P2RX7 is needed for recirculating CD8+ T cell memory, but its role for Trm cells is unclear. Here we showed that P2RX7 supported Trm cell generation by enhancing CD8+ T cell sensing of TGF-ß, which was necessary for tissue residency. P2RX7-deficient Trm cells progressively decayed in non-lymphoid tissues and expressed dysregulated Trm-specific markers. P2RX7 was required for efficient re-expression of the receptor TGF-ßRII through calcineurin signaling. Forced Tgfbr2 expression rescued P2RX7-deficient Trm cell generation, and TGF-ß sensitivity was dictated by P2RX7 agonists and antagonists. Forced Tgfbr2 also rescued P2RX7-deficient Trm cell mitochondrial function. Sustained P2RX7 signaling was required for long-term Trm cell maintenance, indicating that P2RX7 signaling drives induction and CD8+ T cell durability in barrier sites.

USP8 promotes cancer progression and extracellular vesicle-mediated CD8+ T cell exhaustion by deubiquitinating the TGF-ß receptor TßRII.

TGF-ß signaling is a key player in tumor progression and immune evasion, and is associated with poor response to cancer immunotherapies. Here, we identified ubiquitin-specific peptidase 8 (USP8) as a metastasis enhancer and a highly active deubiquitinase in aggressive breast tumors. USP8 acts both as a cancer stemness-promoting factor and an activator of the TGF-ß/SMAD signaling pathway. USP8 directly deubiquitinates and stabilizes the type II TGF-ß receptor TßRII, leading to its increased expression in the plasma membrane and in tumor-derived extracellular vesicles (TEVs). Increased USP8 activity was observed in patients resistant to neoadjuvant chemotherapies. USP8 promotes TGF-ß/SMAD-induced epithelial-mesenchymal transition (EMT), invasion, and metastasis in tumor cells. USP8 expression also enables TßRII+ circulating extracellular vesicles (crEVs) to induce T cell exhaustion and chemoimmunotherapy resistance. Pharmacological inhibition of USP8 antagonizes TGF-ß/SMAD signaling, and reduces TßRII stability and the number of TßRII+ crEVs to prevent CD8+ T cell exhaustion and to reactivate anti-tumor immunity. Our findings not only reveal a novel mechanism whereby USP8 regulates the cancer microenvironment but also demonstrate the therapeutic advantages of engineering USP8 inhibitors to simultaneously suppress metastasis and improve the efficacy of cancer immunotherapy.

Liebig's law of the minimum in the TGF-ß/SMAD pathway.

Cells use signaling pathways to sense and respond to their environments. The transforming growth factor-ß (TGF-ß) pathway produces context-specific responses. Here, we combined modeling and experimental analysis to study the dependence of the output of the TGF-ß pathway on the abundance of signaling molecules in the pathway. We showed that the TGF-ß pathway processes the variation of TGF-ß receptor abundance using Liebig's law of the minimum, meaning that the output-modifying factor is the signaling protein that is most limited, to determine signaling responses across cell types and in single cells. We found that the abundance of either the type I (TGFBR1) or type II (TGFBR2) TGF-ß receptor determined the responses of cancer cell lines, such that the receptor with relatively low abundance dictates the response. Furthermore, nuclear SMAD2 signaling correlated with the abundance of TGF-ß receptor in single cells depending on the relative expression levels of TGFBR1 and TGFBR2. A similar control principle could govern the heterogeneity of signaling responses in other signaling pathways.

Transforming Growth Factor-ß1 Regulates Peroxisomal Genes/Proteins via Smad Signaling in Idiopathic Pulmonary Fibrosis Fibroblasts and Transgenic Mouse Models.

Idiopathic pulmonary fibrosis (IPF) is a chronic human disease with persistent destruction of lung parenchyma. Transforming growth factor-ß1 (TGF-ß1) signaling plays a pivotal role in the initiation and pathogenesis of IPF. As shown herein, TGF-ß1 signaling down-regulated not only peroxisome biogenesis but also the metabolism of these organelles in human IPF fibroblasts. In vitro cell culture observations in human fibroblasts and human lung tissue indicated that peroxisomal biogenesis and metabolic proteins were significantly down-regulated in the lung of 1-month-old transgenic mice expressing a constitutively active TGF-ß type I receptor kinase (ALK5). The peroxisome biogenesis protein peroxisomal membrane protein Pex13p (PEX13p) as well as the peroxisomal lipid metabolic enzyme peroxisomal acyl-coenzyme A oxidase 1 (ACOX1) and antioxidative enzyme catalase were highly up-regulated in TGF-ß type II receptor and Smad3 knockout mice. This study reports a novel mechanism of peroxisome biogenesis and metabolic regulation via TGF-ß1-Smad signaling: interaction of the Smad3 transcription factor with the PEX13 gene in chromatin immunoprecipitation-on-chip assay as well as in a bleomycin-induced pulmonary fibrosis model applied to TGF-ß type II receptor knockout mice. Taken together, data from this study suggest that TGF-ß1 participates in regulation of peroxisomal biogenesis and metabolism via Smad-dependent signaling, opening up novel strategies for the development of therapeutic approaches to inhibit progression of pulmonary fibrosis patients with IPF.

CircRNA CDR1as affects functional repair after spinal cord injury and regulates fibrosis through the SMAD pathway.

Spinal cord injury (SCI) is a complex problem in modern medicine. Fibroblast activation and fibroscarring after SCI impede nerve recovery. Non-coding RNA plays an important role in the progression of many diseases, but the study of its role in the progression of spinal fibrosis is still emerging. Here, we investigated the function of circular RNAs, specifically antisense to the cerebellar degeneration-related protein 1 (CDR1as), in spinal fibrosis and characterized its molecular mechanism and pathophysiology. The presence of CDR1as in the spinal cord was verified by sequencing and RNA expression assays. The effects of inhibition of CDR1as on scar formation, inflammation and nerve regeneration after spinal cord injury were investigated in vivo and in vitro. Further, gene expression of miR-7a-5p and protein expression of transforming Growth Factor Beta Receptor II (TGF-ßR2) were measured to evaluate their predicted interactions with CDR1as. The regulatory effects and activation pathways were subsequently verified by miR-7a-5p inhibitor and siCDR1as. These results indicate that CDR1as/miR-7a-5p/TGF-ßR2 interactions may exert scars and nerves functions and suggest potential therapeutic targets for treating spinal fibrotic diseases.

Engineering Cancer Antigen-Specific T Cells to Overcome the Immunosuppressive Effects of TGF-ß.

Adoptive T cell therapy with T cells expressing affinity-enhanced TCRs has shown promising results in phase 1/2 clinical trials for solid and hematological tumors. However, depth and durability of responses to adoptive T cell therapy can suffer from an inhibitory tumor microenvironment. A common immune-suppressive agent is TGF-ß, which is secreted by tumor cells and cells recruited to the tumor. We investigated whether human T cells could be engineered to be resistant to inhibition by TGF-ß. Truncating the intracellular signaling domain from TGF-ß receptor (TGFßR) II produces a dominant-negative receptor (dnTGFßRII) that dimerizes with endogenous TGFßRI to form a receptor that can bind TGF-ß but cannot signal. We previously generated specific peptide enhanced affinity receptor TCRs recognizing the HLA-A*02-restricted peptides New York esophageal squamous cell carcinoma 1 (NY-ESO-1)157-165/l-Ag family member-1A (TCR: GSK3377794, formerly NY-ESO-1c259) and melanoma Ag gene A10254-262 (TCR: ADP-A2M10, formerly melanoma Ag gene A10c796). In this article, we show that exogenous TGF-ß inhibited in vitro proliferation and effector functions of human T cells expressing these first-generation high-affinity TCRs, whereas inhibition was reduced or abolished in the case of second-generation TCRs coexpressed with dnTGFßRII (e.g., GSK3845097). TGF-ß isoforms and a panel of TGF-ß-associated genes are overexpressed in a range of cancer indications in which NY-ESO-1 is commonly expressed, particularly in synovial sarcoma. As an example, immunohistochemistry/RNAscope identified TGF-ß-positive cells close to T cells in tumor nests and stroma, which had low frequencies of cells expressing IFN-γ in a non-small cell lung cancer setting. Coexpression of dnTGFßRII may therefore improve the efficacy of TCR-transduced T cells.

Introduction of miR-3613-3p as a regulator of transforming growth factor-ß (TGF-ß) signaling pathway in colorectal cancer.

INTRODUCTION: Colorectal cancer (CRC) is the second common cancer and the fourth major reason of cancer death worldwide. Dysregulation of intracellular pathways, such as TGF-ß/SMAD signaling, contributes to CRC development. MicroRNAs (miRNAs) are post-transcriptional regulators that are involved in CRC pathogenesis. Here, we aimed to investigate the effect of miR-3613-3p on the TGF-ß /SMAD signaling pathway in CRC. METHODS & RESULTS: Bioinformatics analysis suggested that miR-3613-3p is a regulator of TGF-Β signaling downstream genes. Then, miR-3613-3p overexpression was followed by downregulation of TGF-ßR1, TGF-ßR2, and SMAD2 expression levels, detected by RT-qPCR. Additionally, dual luciferase assay supported the direct interaction of miR-3613-3p with 3'UTR sequences of TGF-ßR1 and TGF-ßR2 genes. Furthermore, reduced SMAD3 protein level following the miR-3613-3p overexpression verified its suppressive effect against TGF-ß signaling in HCT-116 cells, detected by western blot analysis. Finally, miR-3613-3p overexpression induced sub-G1 arrest in HCT116 cells, detected by flow cytometry, and promoted downregulation of cyclin D1 protein expression, which was detected by western blotting analysis. CONCLUSION: Our findings indicated that miR-3613-3p plays an important role in CRC by targeting the TGF-ß/SMAD signaling pathway and could be considered as a new candidate for further therapy investigations.

MicroRNA-148b secreted by bovine oviductal extracellular vesicles enhance embryo quality through BPM/TGF-beta pathway.

BACKGROUND: Extracellular vesicles (EVs) and their cargoes, including MicroRNAs (miRNAs) play a crucial role in cell-to-cell communication. We previously demonstrated the upregulation of bta-mir-148b in EVs from oviductal fluid of cyclic cows. This miRNA is linked to the TGF-ß pathway in the cell proliferation. Our aim was to verify whether miR-148b is taken up by embryos through gymnosis, validate its target genes, and investigate the effect of miR-148b supplementation on early embryo development and quality. METHODS: Zygotes were cultured in SOF + 0.3% BSA (Control) or supplemented with: 1 µM miR-148b mimics during: D1-D7 (miR148b) or D1-D4 (miR148b-OV: representing miRNA effect in the oviduct) or D4-D7 (miR148b-UT: representing miRNA effect in the uterus) or 1 µM control mimics was used during: D1-D7 (CMimic). Embryos at ≥ 16-cells and D7 blastocysts (BD7) were collected to examine the mRNA abundance of transcripts linked to the TGF-ß pathway (TGFBR2, SMAD1, SMAD2, SMAD3, SMAD5, BMPR2, RPS6KB1, POU5F1, NANOG), total cell number (TC), trophectoderm (TE), and inner cell mass (ICM) were also evaluated. One-way ANOVA was used for all analyses. RESULTS: We demonstrated that miR-148b can be taken up in both 16-cell embryos and BD7 by gymnosis, and we observed a decrease in SMAD5 mRNA, suggesting it's a potential target of miR-148b. Cleavage and blastocysts rates were not affected in any groups; however, supplementation of miR-148b mimics had a positive effect on TC, TE and ICM, with values of 136.4 ± 1.6, 92.5 ± 0.9, 43.9 ± 1.3 for miR148b and 135.3 ± 1.5, 92.6 ± 1.2, 42.7 ± 0.8, for miR148b-OV group. Furthermore, mRNA transcripts of SMAD1 and SMAD5 were decreased (P ≤ 0.001) in 16-cell embryos and BD7 from miR148b and miR148b-OV groups, while POU5F1 and NANOG were upregulated (P ≤ 0.001) in BD7 and TGFBR2 was only downregulated in 16-cell embryos. pSMAD1/5 levels were higher in the miR148b and miR148b-OV groups. CONCLUSIONS: Our findings suggest that supplementation of bta-miR-148b mimics during the entire culture period (D1 - D7) or from D1 - D4 improves embryo quality and influences the TGF-ß signaling pathway by altering the transcription of genes associated with cellular differentiation and proliferation. This highlights the importance of miR-148b on embryo quality and development.

Endophilin A2 protects against renal fibrosis by targeting TGF-ß/Smad signaling.

Renal fibrosis underlies all forms of end-stage kidney disease. Endophilin A2 (EndoA2) plays a role in nephrotic syndrome; however, its effect on renal fibrosis remains unknown. Here, we demonstrate that EndoA2 protects against kidney interstitial fibrosis via the transforming growth factor-ß (TGF-ß)/Smad signaling pathway. Mouse kidneys with fibrosis or kidney biopsy specimens from patients with fibrotic nephropathy had lower levels of EndoA2 protein expression than that in kidneys without fibrosis. In vivo overexpression of EndoA2 with the endophilin A2 transgene (EndoA2Tg ) notably prevented renal fibrosis, decreased the protein expression of profibrotic molecules, suppressed tubular injury, and reduced apoptotic tubular cells in the obstructed kidney cortex of mice with unilateral ureteral obstruction (UUO). In vivo and in vitro overexpression of EndoA2 markedly inhibited UUO- or TGF-ß1-induced phosphorylation of Smad2/3 and tubular epithelial cells dedifferentiation. Furthermore, EndoA2 was co-immunoprecipitated with the type II TGF-ß receptor (TßRII), thus inhibiting the binding of the type I TGF-ß receptor (TßRI) to TßRII. These findings indicate that EndoA2 mitigates renal fibrosis, at least partially, via modulating the TGF-ß/Smad signaling. Targeting EndoA2 may be a new potential therapeutic strategy for treatment of renal fibrosis.

SPARC expression in tumor microenvironment induces partial epithelial-to-mesenchymal transition of esophageal adenocarcinoma cells via cooperating with TGF-ß signaling.

Secreted protein, acidic and rich in cysteine (SPARC) has been characterized as an oncoprotein in esophageal squamous cell carcinoma (ESCC), but its involvement in the pathological development of esophageal adenocarcinoma (ESAD) remains poorly understood. In this study, we aimed to explore the sources of SPARC in the tumor microenvironment (TME) and its functional role in ESAD. Bioinformatic analysis was conducted using data from The Cancer Genome Atlas (TCGA)-esophageal cancer (ESCA) and Genotype-Tissue Expression (GTEx). ESAD tumor cell line OE33 and OE19 cells were used as in vitro cell models. Results showed that SPARC upregulation was associated with unfavorable disease-specific survival (DSS) in ESAD. ESAD tumor cells (OE33 and OE19) had no detectable SPARC protein expression. In contrast, IHC staining in ESAD tumor tissues suggested that peritumoral stromal cells (tumor-associated fibroblasts and macrophages) were the dominant SPARC source in TME. Exogenous SPARC induced partial epithelial-to-mesenchymal transition of ESAD cells, reflected by reduced CDH1 and elevated ZEB1/VIM expression at both mRNA and protein levels. Besides, exogenous SPARC enhanced tumor cell invasion. When TGFBR2 expression was inhibited, the activation of TGF-ß signaling induced by exogenous SPARC was impaired. However, the activating effects were rescued by overexpressing mutant TGFBR2 resistant to the shRNA sequence. Copresence of exogenous SPARC and TGF-ß1 induced higher expression of mesenchymal markers and enhanced the invading capability of ESAD cells than TGF-ß1 alone. In conclusion, this study suggests a potential cross-talk between ESAD tumor stromal cells and cancer cells via a SPARC-TGF-ß1 paracrine network.

Deubiquitinating enzyme USP11 promotes renal tubular cell senescence and fibrosis via inhibiting the ubiquitin degradation of TGF-ß receptor II.

Transforming growth factor-ß1 (TGF-ß1) is regarded as a key factor in promoting renal fibrosis during chronic kidney disease (CKD). Signaling transduction of TGF-ß1 starts with binding to TGF-ß type II receptor (Tgfbr2), a constitutively activated kinase that phosphorylates TGF-ß type I receptor (Tgfbr1), and then activates downstream Smad2/3 or noncanonical pathways. Previous studies show that cellular senescence is associated with the progression of CKD, and accelerated tubular cell senescence is implicated in promoting renal fibrosis. In the present study we investigated the renal parenchymal cell senescence in fibrosis from the sight of posttranslational regulation and focused on Tgfbr2, the important gatekeeper for TGF-ß1 downstream signaling. In mice with unilateral ureteral obstruction (UUO) and folic acid (FA)-induced fibrotic kidneys, we found that Tgfbr2 was markedly elevated without obvious change in its mRNA levels. As an important member of deubiquitinating enzymes, ubiquitin-specific protease 11 (Usp11) was also significantly increased in fibrotic kidneys, and co-distributed with Tgfbr2 in tubular epithelial cells. Pretreatment with Usp11 inhibitor mitoxantrone (MTX, 30 mg · kg-1 · d-1, i.p.) twice a week, for 2 weeks significantly attenuated the elevation of Tgfbr2, activation in downstream senescence-related signaling pathway, as well as renal senescence and fibrosis. In cultured mouse tubular epithelial cells (MTECs), treatment with angiotensin II (Ang-II, 10-7, 10-6 M) dose-dependently elevated both Tgfbr2 and Usp11 levels. Inhibition or knockdown on Usp11 attenuated Ang-II-induced elevation in Tgfbr2 level, and attenuated the activation of downstream senescent-related signaling pathway and as well as cell senescence. We conducted Co-IP experiments, which revealed that Usp11 was able to interact with Tgfbr2, and inhibition of Usp11 increased the ubiquitination of Tgfbr2. Taken together, these results demonstrate that the elevation of Usp11 under pathological condition is implicated in promoting renal fibrosis. Usp11 promotes the development of renal fibrosis by deubiquitinating Tgfbr2, reducing Tgfbr2 ubiquitination degradation, and then facilitating the activation of downstream senescent signaling pathway.

The novel mechanism of Med12-mediated drug resistance in a TGFBR2-independent manner.

Inevitable emergence of drug resistance is the biggest hurdle to both chemotherapies and targeted therapies. Understanding the resistance mechanisms will contribute to identification of biomarkers for predicting response to therapy and design new therapeutic strategies to overcome drug resistance in human cancers. The type II transforming growth factor (TGF)-ß receptor gene (TGFBR2) is frequently frameshift mutated in several cancer types, especially in colorectal, endometrium and gastric cancers cells. Here, we found that Med12, a component of the transcriptional mediator complex, plays a role in modulating chemosensitivity in TGFBR2 deficient cancer cells. Loss of Med12 leads to chemoresistance in multiple TGFBR2 deficient cancer cells. Interestingly, RNA sequencing data revealed that interferon IFN-related DNA damage resistance signature (IRDS) is upregulated in Med12 knockdown cancer cells. And the expression of IRDS pattern is negatively correlated with chemosensitivity. Therefore, our study identifies a novel mechanism of Med12-mediated drug resistance, which is a TGFBR-independent manner.

Oocyte-secreted factor TGFB2 enables mouse cumulus cell expansion in vitro.

Cumulus expansion is necessary for the release of a fertilizable oocyte from the ovary, which is critical for the normal fertilization of mammals. Cumulus expansion requires cooperation between epidermal growth factor (EGF)-like growth factors and oocyte paracrine factors. Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are well-known paracrine factors secreted by oocytes. In addition, transforming growth factor-ß2 (TGFB2) was primarily expressed in oocytes and its membrane receptors type 1 receptor (TGFBR1) and type 2 receptor (TGFBR2) were located in cumulus cells. In our present study, TGFB2 induced expansion of oocytectomized (OOX) complexes and increased the expression of expansion-related genes in the presence of EGF, suggesting that TGFB2 enables cumulus expansion. Inhibition of TGF-ß signaling with SD208 blocked TGFB2-promoted cumulus expansion. Furthermore, in the culture of OOX complexes from mice of Tgfbr2-specific depletion in granulosa cells, TGFB2-promoted cumulus expansion and the expression of expansion-related genes were impaired. These results suggest that TGFB2 could induce cumulus expansion through TGFBR-SMAD2/3 signaling. Tgfb2-specific depletion in oocytes using Zp3-Cre mice had no effect on cumulus expansion in vivo, possibly due to the compensatory effect of other cumulus expansion-enabling factors. Taken together, TGFB2 is involved in expansion-related gene expression and consequent cumulus expansion.

Maresin 1 activates LGR6 signaling to inhibit smooth muscle cell activation and attenuate murine abdominal aortic aneurysm formation.

The specialized pro-resolving lipid mediator maresin 1 (MaR1) is involved in the resolution phase of tissue inflammation. It was hypothesized that exogenous administration of MaR1 would attenuate abdominal aortic aneurysm (AAA) growth in a cytokine-dependent manner via LGR6 receptor signaling and macrophage-dependent efferocytosis of smooth muscle cells (SMCs). AAAs were induced in C57BL/6 wild-type (WT) mice and smooth muscle cell specific TGF-ß2 receptor knockout (SMC-TGFßr2-/- ) mice using a topical elastase AAA model. MaR1 treatment significantly attenuated AAA growth as well as increased aortic SMC α-actin and TGF-ß2 expressions in WT mice, but not SMC-TGFßr2-/- mice, compared to vehicle-treated mice. In vivo inhibition of LGR6 receptors obliterated MaR1-dependent protection in AAA formation and SMC α-actin expression. Furthermore, MaR1 upregulated macrophage-dependent efferocytosis of apoptotic SMCs in murine aortic tissue during AAA formation. In vitro studies demonstrate that MaR1-LGR6 interaction upregulates TGF-ß2 expression and decreases MMP2 activity during crosstalk of macrophage-apoptotic SMCs. In summary, these results demonstrate that MaR1 activates LGR6 receptors to upregulate macrophage-dependent efferocytosis, increases TGF-ß expression, preserves aortic wall remodeling and attenuate AAA formation. Therefore, this study demonstrates the potential of MaR1-LGR6-mediated mitigation of vascular remodeling through increased efferocytosis of apoptotic SMCs via TGF-ß2 to attenuate AAA formation.

Dexamethasone attenuated thoracic aortic aneurysm and dissection in vascular smooth muscle cell Tgfbr2-disrupted mice with CCL8 suppression.

NEW FINDINGS: What is the central question of this study? What is the relationship of chemokine (C-C motif) ligand 8 (CCL8) to thoracic aortic aneurysm and dissection (TAAD) formation in postnatal mice with vascular smooth muscle cell (VSMC) Tgfbr2 disruption, and is dexamethasone a potential treatment? What is the main finding and its importance? CCL8 was associated with the formation of TAAD in VSMC Tgfbr2-disrupted mice. Dexamethasone reduced TAAD formation and inhibited mitogen-activated protein kinase (p-p38) and nuclear factor-κB (p-p65) signalling pathways. CCL8 might be an important promoter of aortic inflammation. Dexamethasone provided potential therapeutic effects in TAAD treatment. ABSTRACT: Aortic inflammation plays a vital role in initiation and progression of thoracic aortic aneurysm and dissection (TAAD). Disturbance of the transforming growth factor-ß (TGF-ß) signalling pathway is believed to be one of the pathogenic mechanisms of TAAD. Initially, Myh11-CreERT2 .Tgfbr2f/f male mice were used to build a TAAD mouse model, and bioinformatic analyses revealed enriched inflammatory signal pathways and upregulated chemokine (C-C motif) ligand 8 (CCL8). So we hypothesized that vascular smooth muscle cell (VSMC) Tgfbr2 disruption in postnatal mice results in aortic inflammation associated with CCL8 secretion. Real-time quantitative PCR and serum enzyme-linked immunosorbent assay (ELISA) results confirmed that CCL8 expression began to increase after VSMC Tgfbr2 disruption. Next, we cultured mouse thoracic aortas ex vivo, and observed that the protein expression of CCL8 in culture supernatants was increased by ELISA. Subsequently, the co-localization of CCL8 with α-smooth muscle actin or CD68 was found to be significantly increased by immunofluorescence. Then, dexamethasone (DEX) was used to treat TAAD in VSMC Tgfbr2-disrupted mice; the results of histochemical, immunofluorescence and immunohistochemical staining indicated that DEX therapy reduced CCL8 secretion, inflammatory cell recruitment, aortic medial thickening, elastic fibre fragmentation, extracellular matrix degradation and contractile apparatus impairment, and thereby ameliorated TAAD formation. Western blotting showed that mitogen-activated protein kinase and nuclear factor-κB signalling pathways in aorta were overactivated after VSMC Tgfbr2 disruption, but inhibited by DEX therapy. Altogether, CCL8 might be an important promoter in TAAD formation of VSMC Tgfbr2-disrupted mice, and DEX provided potential therapeutic effects in TAAD treatment.

Activated Endothelial TGFß1 Signaling Promotes Venous Thrombus Nonresolution in Mice Via Endothelin-1: Potential Role for Chronic Thromboembolic Pulmonary Hypertension.

RATIONALE: Chronic thromboembolic pulmonary hypertension (CTEPH) is characterized by defective thrombus resolution, pulmonary artery obstruction, and vasculopathy. TGFß (transforming growth factor-ß) signaling mutations have been implicated in pulmonary arterial hypertension, whereas the role of TGFß in the pathophysiology of CTEPH is unknown. OBJECTIVE: To determine whether defective TGFß signaling in endothelial cells contributes to thrombus nonresolution and fibrosis. METHODS AND RESULTS: Venous thrombosis was induced by inferior vena cava ligation in mice with genetic deletion of TGFß1 in platelets (Plt.TGFß-KO) or TGFß type II receptors in endothelial cells (End.TGFßRII-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 TGFß1 in platelets did not alter platelet number or function but was associated with faster venous thrombus resolution, whereas endothelial TGFßRII deletion resulted in larger, more fibrotic and higher vascularized venous thrombi. Increased circulating active TGFß1 levels, endothelial TGFßRI/ALK1 (activin receptor-like kinase), and TGFßRI/ALK5 expression were detected in End.TGFßRII-KO mice, and activated TGFß signaling was present in vessel-rich areas of CTEPH specimens. CTEPH-endothelial cells and murine endothelial cells lacking TGFßRII simultaneously expressed endothelial and mesenchymal markers and transcription factors regulating endothelial-to-mesenchymal transition, similar to TGFß1-stimulated endothelial cells. Mechanistically, increased endothelin-1 levels were detected in TGFßRII-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 TGFß1-exposed human and murine endothelial cells and improved venous thrombus resolution and pulmonary vaso-occlusions in End.TGFßRII-KO mice. CONCLUSIONS: Endothelial TGFß1 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.

Upregulation of TGF-ß type II receptor in high glucose-induced vascular smooth muscle cells.

BACKGROUND: Mortality in patients with diabetes mellitus is estimated above 65% due to cardiovascular diseases. The aim of study was to investigate the effects of high-glucose conditions on TGF-ß type II receptor (TGFBR2) expression levels, cell viability, and migration rate in vascular smooth muscle cells (VSMCs). METHODS: VSMCs were incubated in 30 mM and 50 mM of glucose for 24 h, 48 h, and 72 h periods. The gene and protein expression levels were investigated by Real-time qRT-PCR and western blotting techniques, respectively. The cell viability was evaluated by MTT assay. VSMC migration rate was also studied by wound healing assay. RESULTS: The TGFBR2 gene and protein expression levels were significantly upregulated in all the groups treated with glucose in 24 h, 48 h, and 72 h periods. The cell viability was not significantly affected in values of 30 mM and 50 mM of glucose. The increase of migration rate of VSMCs was not significant. CONCLUSION: The results suggested the increased expression levels of TGFBR2 in the response to high glucose conditions may modulate the cellular events through the signaling pathway network in VSMCs.

Bifunctional TGF-ß trap/IL-15 protein complex elicits potent NK cell and CD8+ T cell immunity against solid tumors.

Advances in immunostimulatory and anti-immunosuppressive therapeutics have revolutionized cancer treatment. However, novel immunotherapeutics with these dual functions are not frequently reported. Here we describe the creation of a heterodimeric bifunctional fusion molecule, HCW9218, constructed using our soluble tissue factor (TF)-based scaffold technology. This complex comprises extracellular domains of the human transforming growth factor-ß (TGF-ß) receptor II and a human interleukin-15 (IL-15)/IL-15 receptor α complex. HCW9218 can be readily expressed in CHO cells and purified using antibody-based affinity chromatography in a large-scale manufacturing setting. HCW9218 potently activates mouse natural killer (NK) cells and CD8+ T cells in vitro and in vivo to enhance cell proliferation, metabolism, and antitumor cytotoxic activities. Similarly, human immune cells become activated with increased cytotoxicity following incubation with HCW9218. This fusion complex also exhibits TGF-ß neutralizing activity in vitro and sequesters plasma TGF-ß in vivo. In a syngeneic B16F10 melanoma model, HCW9218 displayed strong antitumor activity mediated by NK cells and CD8+ T cells and increased their infiltration into tumors. Repeat-dose subcutaneous administration of HCW9218 was well tolerated by mice, with a half-life sufficient to provide long-lasting biological activity. Thus, HCW9218 may serve as a novel therapeutic to simultaneously provide immunostimulation and lessen immunosuppression associated with tumors.

A novel circ_MACF1/miR-942-5p/TGFBR2 axis regulates the functional behaviors and drug sensitivity in gefitinib-resistant non-small cell lung cancer cells.

BACKGROUND: Resistance to gefitinib remains a major obstacle for the successful treatment of non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutations. In this paper, we studied the precise actions of circular RNA (circRNA) microtubule actin crosslinking factor 1 (circ_MACF1) in gefitinib resistance. METHODS: We established gefitinib-resistant NSCLC cells (PC9/GR and A549/GR). The levels of circ_MACF1, microRNA (miR)-942-5p, and transforming growth factor beta receptor 2 (TGFBR2) were gauged by quantitative real-time PCR (qRT-PCR) or western blot. Subcellular fractionation and Ribonuclease R (RNase R) assays were done to characterize circ_MACF1. Cell survival, proliferation, colony formation, apoptosis, migration, and invasion were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 5-Ethynyl-2'-Deoxyuridine (EdU), colony formation, flow cytometry, and transwell assays, respectively. Dual-luciferase reporter assays were used to verify the direct relationship between miR-942-5p and circ_MACF1 or TGFBR2. The xenograft assays were used to assess the role of circ_MACF1 in vivo. RESULTS: Circ_MACF1 was down-regulated in A549/GR and PC9/GR cells. Overexpression of circ_MACF1 repressed proliferation, migration, invasion, and promoted apoptosis and gefitinib sensitivity of A549/GR and PC9/GR cells in vitro, as well as inhibited tumor growth under gefitinib in vivo. Circ_MACF1 directly targeted miR-942-5p, and miR-942-5p mediated the regulatory effects of circ_MACF1. TGFBR2 was identified as a direct and functional target of miR-942-5p. Circ_MACF1 modulated TGFBR2 expression through miR-942-5p. CONCLUSION: Our findings demonstrated that circ_MACF1 regulated cell functional behaviors and gefitinib sensitivity of A549/GR and PC9/GR cells at least partially by targeting miR-942-5p to induce TGFBR2 expression.

Microglial CD74 Expression Is Regulated by TGFß Signaling.

Microglia play important roles during physiological and pathological situations in the CNS. Several reports have described the expression of Cd74 in disease-associated and aged microglia. Here, we demonstrated that TGFß1 controled the expression of Cd74 in microglia in vitro and in vivo. Using BV2 cells, primary microglia cultures as well as Cx3cr1CreERT2:R26-YFP:Tgfbr2fl/fl in combination with qPCR, flow cytometry, and immunohistochemistry, we were able to provide evidence that TGFß1 inhibited LPS-induced upregulation of Cd74 in microglia. Interestingly, TGFß1 alone was able to mediate downregulation of CD74 in vitro. Moreover, silencing of TGFß signaling in vivo resulted in marked upregulation of CD74, further underlining the importance of microglial TGFß signaling during regulation of microglia activation. Taken together, our data indicated that CD74 is a marker for activated microglia and further demonstrated that microglial TGFß signaling is important for regulation of Cd74 expression during microglia activation.