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TGF-β1/Smads is a classic signaling pathway, which plays important roles in the development process of organisms. Black porgy Acanthopagrus schlegelii and red porgy Pagrus major are valuable economic fishes, and their hybrid offspring show excellent heterosis traits. Yet the molecular regulation mechanism of the heterosis traits is less clear. Here, we explored the TGF-β1/Smads pathway’s molecular genetic information for heterosis in A. schlegelii ♂ × P. major ♀ (AP) and A. schlegelii ♀ × P. major ♂ (PA) in terms of growth and development. The mRNA expression levels of TGF-β1, TβR-I, TβR-II, and Smad2 genes in different developmental stages of A. schlegelii were detected. Furthermore, the expression levels of TGF-β1, TβR-I, TβR-II, and Smad2 genes in different tissues of adult (mRNA level) and larva (mRNA and protein level) of A. schlegelii, P. major, and their hybrids were determined by both real-time quantitative PCR and Western blot techniques. The results indicated the ubiquitous expression of these genes in all developmental stages of A. schlegelii and in all tested tissues of A. schlegelii, P. major, and its hybrids. Among them, the mRNA of TGF-β1, TβR-I, and TβR-II genes is highly expressed in the liver, gill, kidney, and muscle of black porgy, red porgy, and their hybrid offspring. There are significant changes in gene and protein expression levels in hybrid offspring, which indirectly reflect hybrid advantage. In addition, there was no correlation between protein and mRNA expression levels of Smad2 protein. The results provide novel data for the differential expression of growth and development genes between the reciprocal hybridization generation of black porgy and red porgy and its parents, which is conducive to further explaining the molecular regulation mechanism of heterosis in the growth and development of hybrid porgy.

Cancer immunotherapies have shown clinical success in various types of tumors but the patient response rate is low, particularly in breast cancer. Here we report that malignant breast cancer cells can transfer active TGF-β type II receptor (TβRII) via tumor-derived extracellular vesicles (TEV) and thereby stimulate TGF-β signaling in recipient cells. Up-take of extracellular vesicle-TβRII (EV-TβRII) in low-grade tumor cells initiates epithelial-to-mesenchymal transition (EMT), thus reinforcing cancer stemness and increasing metastasis in intracardial xenograft and orthotopic transplantation models. EV-TβRII delivered as cargo to CD8 + T cells induces the activation of SMAD3 which we demonstrated to associate and cooperate with TCF1 transcription factor to impose CD8 + T cell exhaustion, resulting in failure of immunotherapy. The levels of TβRII + circulating extracellular vesicles (crEV) appears to correlate with tumor burden, metastasis and patient survival, thereby serve as a non-invasive screening tool to detect malignant breast tumor stages. Thus, our findings not only identify a possible mechanism by which breast cancer cells can promote T cell exhaustion and dampen host anti-tumor immunity, but may also identify a target for immune therapy against the most devastating breast tumors. Understanding the factors that hamper immune therapy in breast cancer may increase the range of patients who benefit. Here authors show that breast cancer cells produce and subsequently transfer active TGF-β type II receptors to CD8 + T cells to render them exhausted, thus paralyzing the anti-tumor immune response.

The ALK5 (TGFBR1) receptor serine/threonine kinase transduces TGF-β (Transforming Growth Factor beta) signaling to activate SMAD2/3-dependent and -independent pathways. Here, we aimed to determine the functional consequences of ALK5 mutations in human endometrial cancer (EC). Somatic mutation data were retrieved from publicly available databases. Using seven in silico algorithms, 78.5% (11 of 14) of ALK5 kinase domain mutations in EC, including A230V-ALK5, were predicted to impact protein function. For in vitro studies, we focused on A230V-ALK5 because it was the only mutated residue located within the ATP-binding pocket, which is an important region for both ATP-binding and binding of ATP-competitive inhibitors. Constructs expressing wildtype-, constitutively-active-, kinase-dead-, or mutant A230V-ALK5, were transfected into NIH/3T3 cells. Following TGF-β1 stimulation, transient exogenous expression of A230V-ALK5 resulted in attenuated SMAD2/3 signal transduction and reduced AKT activation. We further showed that the A230V-ALK5 mutant had reduced stability resulting from increased ubiquitin-dependent protein degradation. Our structural modeling predicted that SB-431542, a small molecule ATP-competitive inhibitor of ALK5, binds to the A230V-ALK5 mutant with reduced affinity compared to wildtype-ALK5. We therefore examined the inhibitory effect of SB-431542 and galunisertib on wildtype- and mutant-ALK5 activity using a Smad-binding element (SBE) luciferase reporter assay combined with TGF-β1 stimulation, in NIH/3T3 cells and HEC-265 EC cells. SBE luciferase activity in A230V-ALK5 transfected cells was inhibited less by SB-431542 and galunisertib than in wildtype-ALK5 transfected cells indicating that A230V-ALK5 is less sensitive to inhibition by these agents than wildtype-ALK5, potentially due to changes in SB-431542/A230V-ALK5 binding affinity. Our findings are novel and show that A230V-ALK5 is a partial loss-of-function mutant that attenuates TGF-β1 signal transduction and has reduced sensitivity to ALK5 small molecule inhibitors.

Introduction Tyrosine kinase inhibitors (TKI) are medications of interest in the treatment of Systemic Sclerosis (SSc) because of their ability to inhibit pathways involved in fibrosis. In this open-label pilot trial, our objectives were to assess the safety, efficacy, and molecular change associated with treatment of patients with diffuse cutaneous (dc)SSc with the TKI nilotinib (Tasigna™). Methods Ten adult patients with early dcSSc were treated with nilotinib. Primary endpoints were safety and change in modified Rodnan Skin Score (MRSS) after 6 months. Lesional skin biopsies at baseline, 6 and 12 months of treatment were assessed by histopathology, immunohistochemistry, and DNA microarray. Results Patients had early and active dcSSc with median disease duration of 0.7 years (range 0.5, 1.7) and increasing MRSS in the month prior to baseline (mean +2.9, p=0.02). Seven out of ten patients completed 6 and 12 months of treatment. Seventy-one adverse events (AEs) including 2 serious AEs were observed, and 92 % of AEs were grade 1-2. Two patients discontinued the medication due to mild QTc prolongation. MRSS improved by a mean of 4.2 points (16 %) at 6 months and by 6.3 points (23 %) at 12 months in the 7 completers, p=0.02 and 0.01, respectively. Patients with a decrease in MRSS >20 % from baseline at 12 months (classified as improvers) had significantly higher expression of transforming growth factor beta receptor ( TGFBR ) and platelet-derived growth factor receptor beta ( PDGFRB ) signaling genes at baseline than non-improvers, and the expression of these genes significantly decreased in improvers post-treatment. Conclusion Nilotinib was well tolerated by the majority of patients in this study, with tolerability limited primarily by mild QTc-prolongation. Significant MRSS improvement was observed in these early, active patients, but is not conclusive of treatment effect given the open-label study-design and small number of patients in this pilot study. Improvers had higher levels of expression of genes associated with TGFBR and PDGFRB signaling at baseline, and a significant decrease in the expression of these genes occurred only in patients with higher MRSS improvement. The findings of this pilot study warrant more conclusive evaluation. Trial registration Clinicaltrials.gov NCT01166139 , July 1, 2010.

From sponges to mammals, TGFβ signalling is a central regulator of body plan, cell fate and tissue homeostasis, with receptor architecture highly conserved across metazoans. Here we identify unexpected evolutionary divergence within a key receptor structure: 12 independent ligand-binding domain (LBD) duplications across three receptor classes (ACVR1, BMPR2 and TGFBR2) in diverse vertebrate lineages, including fish, amphibians, birds, and mammals. These duplications reveal previously unrecognized structural and functional plasticity arising from domain-level innovation, including in established model organisms such as zebrafish, African clawed frog and chicken. Recently diverged lineages conserve the membrane-distal LBD and ligand-interacting residues, correlating with enhanced ligand binding, whereas more ancient lineages exhibit elevated evolutionary rates of the membrane-distal LBD associated with inhibitory function. Our findings reveal LBD multimerization as a recurring, lineage-independent mechanism that diversifies and fine-tunes TGFβ signalling, adding a regulatory dimension to one of the best-examined conserved and essential pathways in metazoan biology. Here they show that despite conserved TGFβ signalling, repeated receptor domain multiplications across vertebrates reshape ligand binding and function, revealing unexpected structural plasticity in a core pathway.

The transforming growth factor-β (TGF-β) superfamily encodes a large group of proteins, including TGF-β isoforms, bone morphogenetic proteins and activins that act through conserved cell-surface receptors and signaling co-receptors. TGF-β signaling in insects controls physiological events, including growth, development, diapause, caste determination and metamorphosis. In this study, we used the red flour beetle, Tribolium castaneum, as a model species to investigate the role of the type I TGF-β receptor, saxophone (Sax), in mediating development. Developmental and tissue-specific expression profiles indicated Sax is constitutively expressed during development with lower expression in 19- and 20-day (6th instar) larvae. RNAi knockdown of Sax in 19-day larvae prolonged developmental duration from larvae to pupae and significantly decreased pupation and adult eclosion in a dose-dependent manner. At 50 ng dsSax/larva, Sax knockdown led to an 84.4% pupation rate and 46.3% adult emergence rate. At 100 ng and 200 ng dsSax/larva, pupation was down to 75.6% and 50%, respectively, with 0% adult emergence following treatments with both doses. These phenotypes were similar to those following knockdowns of 20-hydroxyecdysone (20E) receptor genes, ecdysone receptor (EcR) or ultraspiracle  protein (USP). Expression of 20E biosynthesis genes disembodied and spookier, 20E receptor genes EcR and USP, and 20E downstream genes BrC and E75, were suppressed after the Sax knockdown. Topical application of 20E on larvae treated with dsSax partially rescued the dsSax-driven defects. We can infer that the TGF-β receptor gene Sax influences larval-pupal-adult development via 20E signaling in T. castaneum.

Cleft lip and cleft palate are among the most common congenital anomalies and are the result of incomplete fusion of embryonic craniofacial processes or palatal shelves, respectively. We know that genetics play a large role in these anomalies but the list of known causal genes is far from complete. As part of a larger sequencing effort of patients with congenital craniofacial anomalies, we identified a rare candidate variant in transforming growth factor beta receptor 2 ( TGFBR2 ). This variant alters a highly conserved amino acid and is predicted to be pathogenic by a number of metrics. The family history and population genetics suggest that this specific variant would be incompletely penetrant, but this gene has been convincingly implicated in craniofacial development. In order to test the hypothesis this might be a causal variant, we used genome editing to create the orthologous variant in a new mouse model. Surprisingly, Tgfbr2 V387M mice did not exhibit craniofacial anomalies or have reduced survival, suggesting Tgfbr2 V387M is not a causal variant for cleft palate/ micrognathia. The discrepancy between in silico predictions and mouse phenotypes highlights the complexity of translating human genetic findings to mouse models. We expect these findings will aid in interpretation of future variants seen in TGFBR2 from ongoing sequencing of patients with congenital craniofacial anomalies.

The transforming growth factor-β (TGFβ) family plays an important role in many developmental processes and when mutated often contributes to various diseases. Marfan syndrome is a genetic disease with an occurrence of approximately 1 in 5,000. The disease is caused by mutations in fibrillin, which lead to an increase in TGFβ ligand activity, resulting in abnormalities of connective tissues which can be life-threatening. Mutations in other components of TGFβ signaling (receptors, Smads, Schnurri) lead to similar diseases with attenuated phenotypes relative to Marfan syndrome. In particular, mutations in TGFβ receptors, most of which are clustered at the C-terminal end, result in Marfan-like (MFS-like) syndromes. Even though it was assumed that many of these receptor mutations would reduce or eliminate signaling, in many cases signaling is active. From our previous studies on receptor trafficking in C. elegans, we noticed that many of these receptor mutations that lead to Marfan-like syndromes overlap with mutations that cause mis-trafficking of the receptor, suggesting a link between Marfan-like syndromes and TGFβ receptor trafficking. To test this hypothesis, we introduced three of these key MFS and MFS-like mutations into the C. elegans TGFβ receptor and asked if receptor trafficking is altered. We find that in every case studied, mutated receptors mislocalize to the apical surface rather than basolateral surface of the polarized intestinal cells. Further, we find that these mutations result in longer animals, a phenotype due to over-stimulation of the nematode TGFβ pathway and, importantly, indicating that function of the receptor is not abrogated in these mutants. Our nematode models of Marfan syndrome suggest that MFS and MFS-like mutations in the type II receptor lead to mis-trafficking of the receptor and possibly provides an explanation for the disease, a phenomenon which might also occur in some cancers that possess the same mutations within the type II receptor (e.g. colon cancer).

The TGF signaling pathway is a key regulator of cancer progression. In this work, we report for the first time the antitumor activity of TβRII-SE/Fc, a novel peptibody whose targeting domain is comprised of the soluble endogenous isoform of the human TGF-β type II receptor (TβRII-SE). Overexpression of TβRIISE/Fc reduces in vitro cell proliferation and migration while inducing cell cycle arrest and apoptosis in human colorectal cancer-derived cell lines. Moreover, TβRII-SE/Fc overexpression reduces tumorigenicity in BALB/c nude athymic mice. Our results revealed that TRII-SE/Fc-expressing tumors were significantly reduced in size or were even incapable of developing. We also demonstrated that the novel peptibody has the ability to inhibit the canonical TGF-β and BMP signaling pathways while identifying SMAD-dependent and independent proteins involved in tumor progression that are modulated by TβRII-SE/Fc. These findings provide insights into the underlying mechanism responsible for the antitumor activity of TβRII-SE/Fc. Although more studies are required to demonstrate the effectiveness and safety of the novel peptibody as a new therapeutic for the treatment of cancer, our initial in vitro and in vivo results in human colorectal tumor-derived cell lines are highly encouraging. Our results may serve as the foundation for further research and development of a novel biopharmaceutical for oncology.

Microglial phagocytosis exerts essential roles in neurodegeneration, but how phagocytic processes may reciprocally regulate microglia remains incompletely understood. Here, we report that microglial response in the mouse model of pathological axonal degeneration depends on the phagocytic receptor MerTK. The MerTK-triggered downstream phospholipase C signal is sufficient to induce the up-regulation of PU.1 and IRF8, the two central transcription factors governing microglial functions. Chromatin immunoprecipitation-sequencing analyses identify that PU.1 and IRF8 directly target the gene locus of TGFβ1, and disruption of this PU.1-IRF8 targeting site abolishes the induction of microglial TGFβ1 during neurodegeneration. Of importance, neurodegeneration-induced TGFβ1 acts in an autocrine manner, and the microglia-specific deletion of TGFβ1 or its receptors TGFβR1 or TGFβR2 blocks microglial response. Moreover, microglial TGFβ1 autocrine signaling similarly occurs in the 5×FAD mouse model of Alzheimer’s disease and in human patients. These results have delineated an important mechanism underlying microglial response to neurodegeneration. The interplay between microglial phagocytosis and function is not completely understood. Here, the authors show that in response to neurodegeneration, the phagocytic receptor MerTK in microglia triggers PU.1 and IRF8, which elicit TGFβ1 as an autocrine signal. This signaling pathway also occurs in Alzheimer’s disease mouse model and patient cells.