Oridonin suppresses gastric cancer SGC-7901 cell proliferation by targeting the TNF-alpha/androgen receptor/TGF-beta signalling pathway axis.

Statistics provided by GLOBOCAN list gastric cancer as the sixth most common, with a mortality ranking of third highest for the year 2020. In China, a herb called Rabdosia rubescens (Hemsl.) H.Hara, has been used by local residents for the treatment of digestive tract cancer for hundreds of years. Oridonin, the main ingredient of the herb, has a curative effect for gastric cancer, but the mechanism has not been previously clarified. This study mainly aimed to investigate the role of TNF-alpha/Androgen receptor/TGF-beta signalling pathway axis in mediating the proliferation inhibition of oridonin on gastric cancer SGC-7901 cells. MTT assay, cell morphology observation assay and fluorescence assay were adopted to study the efficacy of oridonin on cell proliferation. The network pharmacology was used to predict the pathway axis regulated by oridonin. Western blot assay was adopted to verify the TNF-α/Androgen receptor/TGF-ß signalling pathway axis regulation on gastric cancer by oridonin. The results showed Oridonin could inhibit the proliferation of gastric cancer cells, change cell morphology and cause cell nuclear fragmentation. A total of 11signaling pathways were annotated by the network pharmacology, among them, Tumour necrosis factor alpha (TNF-α) signalling pathway, androgen receptor (AR) signalling pathway and transforming growth factor (TGF-ß) signalling pathway account for the largest proportion. Oridonin can regulate the protein expression of the three signalling pathways, which is consistent with the results predicted by network pharmacology. These findings indicated that oridonin can inhibit the proliferation of gastric cancer SGC-7901 cells by regulating the TNF-α /AR /TGF-ß signalling pathway axis.

Detrimental role of SIX1 in hepatic lipogenesis and fibrosis of non-alcoholic fatty liver disease.

BACKGROUND AND AIMS: Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide. Aberrant lipid metabolism and accumulation of extracellular matrix proteins are hallmarks of the disease, but the underlying mechanisms are largely unknown. This study aims to elucidate the key role of sine oculis homeobox homologue 1 (SIX1) in the development of NAFLD. METHODS: Alb-Cre mice were administered the AAV9 vector for SIX1 liver-specific overexpression or knockdown. Metabolic disorders, hepatic steatosis, and inflammation were monitored in mice fed with HFHC or MCD diet. High throughput CUT&Tag analysis was employed to investigate the mechanism of SIX1 in diet-induced steatohepatitis. RESULTS: Here, we found increased SIX1 expression in the livers of NAFLD patients and animal models. Liver-specific overexpression of SIX1 using adeno-associated virus serotype 9 (AAV9) provoked more severe inflammation, metabolic disorders, and hepatic steatosis in the HFHC or MCD-induced mice model. Mechanistically, we demonstrated that SIX1 directly activated the expression of liver X receptor α (LXRα) and liver X receptor ß (LXRß), thus inducing de novo lipogenesis (DNL). In addition, our results also illustrated a critical role of SIX1 in regulating the TGF-ß pathway by increasing the levels of type I and II TGF-ß receptor (TGFßRI/TGFßRII) in hepatic stellate cells (HSCs). Finally, we found that liver-specific SIX1 deficiency could ameliorate diet-induced NAFLD pathogenesis. CONCLUSION: Our findings suggest a detrimental function of SIX1 in the progression of NAFLD. The direct regulation of LXRα/ß and TGF-ß signalling by SIX1 provides a new regulatory mechanism in hepatic steatosis and fibrosis.

Myhre syndrome: expanding its paediatric phenotypic spectrum.

Myhre syndrome is a rare disease secondary to pathogenic variants in SMAD4 gene. It is a multisystem disease characterised by short stature, deafness, joint stiffness, craniofacial dysmorphism, and potential cardiac manifestations. Herein, we report two new paediatric cases of Myhre syndrome who, additionally, presented with mid-aortic syndrome. This confirms and extends the scarce reports describing the association between these two entities.

The multifunctional adaptor protein HIP-55 couples Smad7 to accelerate TGF-ß type I receptor degradation.

Transforming growth factor ß (TGF-ß) is a multifunctional polypeptide that plays critical roles in regulating a broad range of cellular functions and physiological processes. TGF-ß signalling dysfunction contributes to many disorders, such as cardiovascular diseases, cancer and immunological diseases. The homoeostasis of negative feedback regulation is critical for signal robustness, duration and specificity, which precisely control physiological and pathophysiological processes. However, the underlying mechanism by which the negative regulation of TGF-ß signalling is integrated and coordinated is still unclear. Here, we reveal that haematopoietic progenitor kinase-interacting protein of 55 kDa (HIP-55) was upregulated upon TGF-ß stimulation, while the loss of HIP-55 caused TGF-ß signalling overactivation and the abnormal accumulation of downstream extracellular matrix (ECM) genes. HIP-55 interacts with Smad7 and competes with Smad7/Axin complex formation to inhibit the Axin-mediated degradation of Smad7. HIP-55 further couples Smad7 to TßRI but not TßRII, driving TßRI degradation. Altogether, our findings demonstrate a new mechanism by which the effector and negative feedback functions of HIP-55 are coupled and may provide novel strategies for the treatment of TGF-ß signalling-related human diseases.

SLC39A5 dysfunction impairs extracellular matrix synthesis in high myopia pathogenesis.

High myopia is one of the leading causes of visual impairment worldwide with high heritability. We have previously identified the genetic contribution of SLC39A5 to nonsyndromic high myopia and demonstrated that disease-related mutations of SLC39A5 dysregulate the TGF-ß pathway. In this study, the mechanisms underlying SLC39A5 involvement in the pathogenesis of high myopia are determined. We observed the morphogenesis and migration abnormalities of the SLC39A5 knockout (KO) human embryonic kidney cells (HEK293) and found a significant injury of ECM constituents. RNA-seq and qRT-PCR revealed the transcription decrease in COL1A1, COL2A1, COL4A1, FN1 and LAMA1 in the KO cells. Further, we demonstrated that TGF-ß signalling, the regulator of ECM, was inhibited in SLC39A5 depletion situation, wherein the activation of receptor Smads (R-Smads) via phosphorylation was greatly blocked. SLC39A5 re-expression reversed the phenotype of TGF-ß signalling and ECM synthesis in the KO cells. The fact that TGF-ß signalling was zinc-regulated and that SLC39A5 was identified as a zinc transporter urged us to check the involvement of intracellular zinc in TGF-ß signalling impairment. Finally, we determined that insufficient zinc chelation destabilized Smad proteins, which naturally inhibited TGF-ß signalling. Overall, the SLC39A5 depletion-induced zinc deficiency destabilized Smad proteins, which inhibited the TGF-ß signalling and downstream ECM synthesis, thus contributing to the pathogenesis of high myopia. This discovery provides a deep insight into myopic development.

TGF-ß isoforms inhibit hepatitis C virus propagation in transforming growth factor beta/SMAD protein signalling pathway dependent and independent manners.

Transforming growth factor beta (TGF-ß) plays an important role in the viral liver disease progression via controlling viral propagation and mediating inflammation-associated responses. However, the antiviral activities and mechanisms of TGF-ß isoforms, including TGF-ß1, TGF-ß2 and TGF-ß3, remain unclear. Here, we demonstrated that all of the three TGF-ß isoforms were increased in Huh7.5 cells infected by hepatitis C virus (HCV), but in turn, the elevated TGF-ß isoforms could inhibit HCV propagation with different potency in infectious HCV cell culture system. TGF-ß isoforms suppressed HCV propagation through interrupting several different stages in the whole HCV life cycle, including virus entry and intracellular replication, in TGF-ß/SMAD signalling pathway-dependent and TGF-ß/SMAD signalling pathway-independent manners. TGF-ß isoforms showed additional anti-HCV activities when combined with each other. However, the elevated TGF-ß1 and TGF-ß2, not TGF-ß3, could also induce liver fibrosis with a high expression of type I collagen alpha-1 and α-smooth muscle actin in LX-2 cells. Our results showed a new insight into TGF-ß isoforms in the HCV-related liver disease progression.

TGF-ßR inhibitor SB431542 restores immune suppression induced by regulatory B-T cell axis and decreases tumour burden in murine fibrosarcoma.

The contribution of immune cells in soft tissue sarcomas (STS) is not completely known and understanding their role is very essential for employing immunotherapy strategies. Here, we show that murine fibrosarcoma-conditioned medium promoted total spleen cell proliferation but inhibited T cell responses to mitogenic and allo-antigen-mediated stimulation. This increased proliferation was found to be in B cells resulting in generation of Breg further leading to Treg population. This was found to be the same in vitro and in vivo. The phenotype of these B cells was CD19+CD81+CD27+CD25+PD-L1hi and they secreted both IL-10 and TGF-ß. These tumor evoked Bregs (tBreg), when co-cultured with B depleted T cells, suppressed their proliferation in response to anti-CD3/CD28 stimulation. tBreg-induced suppression of T cell responses was not abrogated by the inhibition or neutralization of IL-10 but by the small molecule inhibitor of TGFß Receptor type I, SB431542. While SB531542 per se was not cytotoxic to tumor cells, administration of SB431542 in tumor-bearing mice (TBM) significantly reduced the tumor burden. In addition, the treatment significantly reduced Treg cells and rescued proliferation of T cells in response to mitogen and allo-antigen. Collectively, our results identify that tumor evoked Breg cells mediate T cell immune suppression through TGFß-mediated pathway and that targeting the Breg-Treg axis can be potentially used as an immunotherapy agent.

SMA-10 Is a Non-Canonical Member of the TGF-ß Sma/Mab Pathway and Immunity Regulator via the DAF-2 Insulin Receptor in Caenorhabditis elegans.

Transforming growth factor ß (TGF-ß) signalling pathways are highly conserved across metazoa and play essential roles not only during development but also in adult tissue maintenance. Alterations of these pathways usually result in a plethora of pathologies. In the nematode Caenorhabditis elegans, the TGF-ß Sma/Mab (small/male abnormal) pathway regulates various worm phenotypes such as body size, immune response, ageing, matricide and reproductive span. SMA-10 has been described as a positive modulator of worm body size through the TGF-ß Sma/Mab pathway. To better understand if SMA-10 is a core component of the pathway, we use gene epistatic analysis to assess the contribution of SMA-10 to various phenotypes regulated by TGF-ß Sma/Mab. We confirm that SMA-10 controls body size and find that it also affects the matricide and reproductive span of the nematodes. However, neither male tail formation (previously reported) nor ageing appeared altered. Lastly, although null sma-10 worms are more susceptible to Pseudomonas aeruginosa infections than wild-types, this response does not depend on TGF-ß Sma/Mab but on the insulin receptor DAF-2. We also show that the expression of sma-10 in either hypodermis or intestine fully rescues the wild-type immune response. Our results contribute to understanding the role of SMA-10 as a context-dependent component of TGF-ß Sma/Mab, and reveal a function of SMA-10 in immunity in association to the Insulin/insulin-like growth factor signalling (IIS) pathway.

Protective mechanism of SIRT1 on Hcy-induced atrial fibrosis mediated by TRPC3.

High plasma levels of homocysteine (Hcy) are regarded as a risk factor for atrial fibrillation (AF), which is closely associated with the pathological consequence of atrial fibrosis and can lead to heart failure with a high mortality rate; here, we show that atrial fibrosis is mediated by the relationship between canonical transient receptor potential 3 (TRPC3) channels and sirtuin type 1 (SIRT1) under the stimulation of Hcy. The left atrial appendage was obtained from patients with either sinus rhythm (SR) or AF and used to evaluate the relationship between the concentration of Hcy and a potential mechanism of cardiac fibrosis mediated by TRPC3 and SIRT1. We next performed transverse aortic constriction (TAC) in mouse to investigate the relationship. The mechanisms underlying atrial fibrosis involving TRPC3 and SIRT1 proteins were explored by co-IP, BLI and lentivirus transfection experiments. qPCR and WB were performed to analyse gene and protein expression, respectively. The higher level of atrial fibrosis was observed in the HH mouse group with a high Hcy diet. Such results suggest that AF patients may be more susceptible to atrial fibrosis and possess a high probability of progressing to hyperhomocysteinemia. Moreover, our findings are consistent with the hypothesis that TRPC3 channel up-regulation leads to abnormal accumulation of collagen, with the down-regulation of SIRT1 as an aetiological factor of high Hcy, which in turn predisposes to atrial fibrosis and strongly enhances the possibility of AF.

Ablation of Gadd45ß ameliorates the inflammation and renal fibrosis caused by unilateral ureteral obstruction.

The growth arrest and DNA damage-inducible beta (Gadd45ß) protein have been associated with various cellular functions, but its role in progressive renal disease is currently unknown. Here, we examined the effect of Gadd45ß deletion on cell proliferation and apoptosis, inflammation, and renal fibrosis in an early chronic kidney disease (CKD) mouse model following unilateral ureteral obstruction (UUO). Wild-type (WT) and Gadd45ß-knockout (KO) mice underwent either a sham operation or UUO and the kidneys were sampled eight days later. A histological assay revealed that ablation of Gadd45ß ameliorated UUO-induced renal injury. Cell proliferation was higher in Gadd45ß KO mouse kidneys, but apoptosis was similar in both genotypes after UUO. Expression of pro-inflammatory cytokines after UUO was down-regulated in the kidneys from Gadd45ß KO mice, whereas UUO-mediated immune cell infiltration remained unchanged. The expression of pro-inflammatory cytokines in response to LPS stimulation decreased in bone marrow-derived macrophages from Gadd45ß KO mice compared with that in WT mice. Importantly, UUO-induced renal fibrosis was ameliorated in Gadd45ß KO mice unlike in WT mice. Gadd45ß was involved in TGF-ß signalling pathway regulation in kidney fibroblasts. Our findings demonstrate that Gadd45ß plays a crucial role in renal injury and may be a therapeutic target for the treatment of CKD.

Role of miR-218-GREM1 axis in epithelial-mesenchymal transition of oral squamous cell carcinoma: An in vivo and vitro study based on microarray data.

Oral squamous cell carcinoma (OSCC) is a prevalent cancer that develops in the head and neck area and has high annual mortality despite optimal treatment. microRNA-218 (miR-218) is a tumour inhibiting non-coding RNA that has been reported to suppress the cell proliferation and invasion in various cancers. Thus, our study aims to determine the mechanism underlying the inhibitory role of miR-218 in OSCC. We conducted a bioinformatics analysis to screen differentially expressed genes in OSCC and their potential upstream miRNAs. After collection of surgical OSCC tissues, we detected GREM1 expression by immunohistochemistry, RT-qPCR and Western blot analysis, and miR-218 expression by RT-qPCR. The target relationship between miR-218 and GREM1 was assessed by dual-luciferase reporter gene assay. After loss- and gain-of-function experiments, OSCC cell proliferation, migration and invasion were determined by MTT assay, scratch test and Transwell assay, respectively. Expression of TGF-ß1, Smad4, p21, E-cadherin, Vimentin and Snail was measured by RT-qPCR and Western blot analysis. Finally, effects of miR-218 and GREM1 on tumour formation and liver metastasis were evaluated in xenograft tumour-bearing nude mice. GREM1 was up-regulated, and miR-218 was down-regulated in OSCC tissues, and GREM1 was confirmed to be the target gene of miR-218. Furthermore, after up-regulating miR-218 or silencing GREM1, OSCC cell proliferation, migration and invasion were reduced. In addition, expression of TGF-ß signalling pathway-related genes was diminished by overexpressing miR-218 or down-regulating GREM1. Finally, up-regulated miR-218 or down-regulated GREM1 reduced tumour growth and liver metastasis in vivo. Taken together, our findings suggest that the overexpression of miR-218 may inhibit OSCC progression by inactivating the GREM1-dependent TGF-ß signalling pathway.

Gastrin-releasing peptide induces fibrotic response in MRC5s and proliferation in A549s.

Idiopathic pulmonary fibrosis (IPF) is a complex lung disease, whose build-up scar tissue is induced by several molecules. Gastrin-releasing peptide (GRP) is released from pulmonary neuroendocrine cells, alveolar macrophages, and some nerve endings in the lung. A possible role of GRP in IPF is unclear. We aimed to investigate the fibrotic response to GRP, at the cellular level in MRC5 and A549 cell lines. The proliferative and fibrotic effects of GRP on these cells were evaluated by using BrdU, immunoblotting, immunofluorescence and qRT-PCR for molecules associated with myofibroblast differentiation, TGF-ß and Wnt signalling. All doses of GRP increased the amount of BrdU incorporation in A549 cells. In contrast, the amount of BrdU increased in MRC5 cells in the first 24 h, though progressively decreased by 72 h. GRP did not stimulate epithelial-mesenchymal transition in A549 cells, rather, it stimulated the differentiation of MRC5 cells into myofibroblasts. Furthermore, GRP induced gene and protein expressions of p-Smad2/3 and Smad4, and reduced the levels of Smad7 in MRC5 cells. In addition, GRP decreased Wnt5a protein levels and stimulated ß-catenin activation by increasing Wnt4, Wnt7a and ß-catenin protein levels. GRP caused myofibroblast differentiation by inducing TGF-ßand Wnt pathways via paracrine and autocrine signalling in MRC5 cells. In conclusion, GRP may lead to pulmonary fibrosis due to its proliferative and fibrotic effects on lung fibroblasts. The abrogation of GRP-mediated signal activation might be considered as a treatment modality for fibrotic lung diseases. Video Abstract.

Deletion of delta-like 1 homologue accelerates fibroblast-myofibroblast differentiation and induces myocardial fibrosis.

AIMS: Myocardial fibrosis is associated with profound changes in ventricular architecture and geometry, resulting in diminished cardiac function. There is currently no information on the role of the delta-like homologue 1 (Dlk1) in the regulation of the fibrotic response. Here, we investigated whether Dlk1 is involved in cardiac fibroblast-to-myofibroblast differentiation and regulates myocardial fibrosis and explored the molecular mechanism underpinning its effects in this process. METHODS AND RESULTS: Using Dlk1-knockout mice and adenoviral gene delivery, we demonstrate that overexpression of Dlk1 in cardio-fibroblasts resulted in inhibition of fibroblast proliferation and differentiation into myofibroblasts. This process is mediated by TGF-ß1 signalling, since isolated fibroblasts lacking Dlk1 exhibited a higher activation of the TGF-ß1/Smad-3 pathway at baseline, leading to an earlier acquisition of a myofibroblast phenotype. Likewise, Dlk1-null mice displayed increased TGF-ß1/Smad3 cardiac activity, resulting in infiltration/accumulation of myofibroblasts, induction and deposition of extra-domain A-fibronectin isoform and collagen, and activation of pro-fibrotic markers. Furthermore, these profibrotic events were associated with disrupted myofibril integrity, myocyte hypertrophy, and cardiac dysfunction. Interestingly, Dlk1 expression was down-regulated in ischaemic human and porcine heart tissues. Mechanistically, miR-370 mediated Dlk1's regulation of cardiac fibroblast-myofibroblast differentiation by directly targeting TGFß-R2/Smad-3 signalling, while the Dlk1 canonical target, Notch pathway, does not seem to play a role in this process. CONCLUSION: These findings are the first to demonstrate an inhibitory role of Dlk1 of cardiac fibroblast-to-myofibroblast differentiation by interfering with TGFß/Smad-3 signalling in the myocardium. Given the deleterious effects of continuous activation of this pathway, we propose Dlk1 as a new potential candidate for therapy in cases where aberrant TGFß signalling leads to chronic fibrosis.

Actin cytoskeleton assembly regulates collagen production via TGF-ß type II receptor in human skin fibroblasts.

The dermal compartment of skin is primarily composed of collagen-rich extracellular matrix (ECM), which is produced by dermal fibroblasts. In Young skin, fibroblasts attach to the ECM through integrins. During ageing, fragmentation of the dermal ECM limits fibroblast attachment. This reduced attachment is associated with decreased collagen production, a major cause of skin thinning and fragility, in the elderly. Fibroblast attachment promotes assembly of the cellular actin cytoskeleton, which generates mechanical forces needed for structural support. The mechanism(s) linking reduced assembly of the actin cytoskeleton to decreased collagen production remains unclear. Here, we report that disassembly of the actin cytoskeleton results in impairment of TGF-ß pathway, which controls collagen production, in dermal fibroblasts. Cytoskeleton disassembly rapidly down-regulates TGF-ß type II receptor (TßRII) levels. This down-regulation leads to reduced activation of downstream effectors Smad2/Smad3 and CCN2, resulting in decreased collagen production. These responses are fully reversible; restoration of actin cytoskeleton assembly up-regulates TßRII, Smad2/Smad3, CCN2 and collagen expression. Finally, actin cytoskeleton-dependent reduction of TßRII is mediated by induction of microRNA 21, a potent inhibitor of TßRII protein expression. Our findings reveal a novel mechanism that links actin cytoskeleton assembly and collagen expression in dermal fibroblasts. This mechanism likely contributes to loss of TßRII and collagen production, which are observed in aged human skin.

Dihydroartemisinin up-regulates VE-cadherin expression in human renal glomerular endothelial cells.

The antimalarial agent dihydroartemisinin (DHA) has been shown to be anti-inflammatory. In this study, we found that DHA increased the expression of the junctional protein vascular endothelial (VE)-cadherin in human renal glomerular endothelial cells. In addition, DHA inhibited TGF-ß RI-Smad2/3 signalling and its downstream effectors SNAIL and SLUG, which repress VE-cadherin gene transcription. Correspondingly, DHA decreased the binding of SNAIL and SLUG to the VE-cadherin promoter. Together, our results suggest an effect of DHA in regulating glomerular permeability by elevation of VE-cadherin expression.

TGF-ß signalling defect is linked to low CD39 expression on regulatory T cells and methotrexate resistance in rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune arthropathy characterized by chronic articular inflammation. Methotrexate (MTX) remains the first-line therapy for RA and its anti-inflammatory effect is associated with the maintenance of high levels of extracellular adenosine (ADO). Nonetheless, up to 40% of RA patients are resistant to MTX treatment and this is linked to a reduction of CD39 expression, an ectoenzyme involved in the generation of extracellular ADO by ATP metabolism, on circulating regulatory T cells (Tregs). However, the mechanism mediating the reduction of CD39 expression on Tregs is unknown. Here we demonstrated that the impairment in TGF-ß signalling lead to the reduction of CD39 expression on Tregs that accounts for MTX resistance. TGF-ß increases CD39 expression on Tregs via the activation of TGFBRII/TGFBRI, SMAD2 and the transcription factor CREB, which is activated in a p38-dependent manner and induces CD39 expression by promoting ENTPD1 gene transcription. Importantly, unresponsive patients to MTX (UR-MTX) show reduced expression of TGFBR2 and CREB1 and decreased levels of p-SMAD2 and p-CREB in Tregs compared to MTX-responsive patients (R-MTX). Furthermore, RA patients carrying at least one mutant allele for rs1431131 (AT or AA) of the TGFBR2 gene are significantly (p = 0.0006) associated with UR-MTX. Therefore, we have uncovered a molecular mechanism for the reduced CD39 expression on Tregs, and revealed potential targets for therapeutic intervention for MTX resistance.

Transforming growth factor-ß-dependent Wnt secretion controls myofibroblast formation and myocardial fibrosis progression in experimental autoimmune myocarditis.

AIMS: Myocardial fibrosis critically contributes to cardiac dysfunction in inflammatory dilated cardiomyopathy (iDCM). Activation of transforming growth factor-ß (TGF-ß) signalling is a key-step in promoting tissue remodelling and fibrosis in iDCM. Downstream mechanisms controlling these processes, remain elusive. METHODS AND RESULTS: Experimental autoimmune myocarditis (EAM) was induced in BALB/c mice with heart-specific antigen and adjuvant. Using heart-inflammatory precursors, as well as mouse and human cardiac fibroblasts, we demonstrated rapid secretion of Wnt proteins and activation of Wnt/ß-catenin pathway in response to TGF-ß signalling. Inactivation of extracellular Wnt with secreted Frizzled-related protein 2 (sFRP2) or inhibition of Wnt secretion with Wnt-C59 prevented TGF-ß-mediated transformation of inflammatory precursors and cardiac fibroblasts into pathogenic myofibroblasts. Inhibition of T-cell factor (TCF)/ß-catenin-mediated transcription with ICG-001 or genetic loss of ß-catenin also prevented TGF-ß-induced myofibroblasts formation. Furthermore, blocking of Smad-independent TGF-ß-activated kinase 1 (TAK1) pathway completely abrogated TGF-ß-induced Wnt secretion. Activation of Wnt pathway in the absence of TGF-ß, however, failed to transform precursors into myofibroblasts. The critical role of Wnt axis for cardiac fibrosis in iDCM is also supported by elevated Wnt-1/Wnt-5a levels in human samples from hearts with myocarditis. Accordingly, and as an in vivo proof of principle, inhibition of Wnt secretion or TCF/ß-catenin-mediated transcription abrogated the development of post-inflammatory fibrosis in EAM. CONCLUSION: We identified TAK1-mediated rapid Wnt protein secretion as a novel downstream key mechanism of TGF-ß-mediated myofibroblast differentiation and myocardial fibrosis progression in human and mouse myocarditis. Thus, pharmacological targeting of Wnts might represent a promising therapeutic approach against iDCM in the future.

TGF-ß and IL-6 family signalling crosstalk: an integrated model.

Mathematical models for TGF-ß and IL-6 signalling have been linked, providing a platform for analyzing the crosstalk between the systems. An integrated IL-6:TGF-ß model was developed via a reduced set of reaction equations which incorporate both feedback loops and appropriate time-delays for transcription and translation processes. The model simulates stable, robust and realistic responses to both ligands. Pulsatile (multiple pulses) inputs for both TGF-ß and IL-6 have been simulated to investigate the effects of each ligand on the sensitivity, equilibrium and dynamic responses of the integrated signalling system. In our simulations the crosstalk between constant IL-6 and TGF-ß signalling via SMAD7 does not appear to be sufficient to render the cells resistant to TGF-ß inhibition. However, the simulations predict that pulsatile IL-6 stimulation would increase SMAD7 levels substantially and consequentially, lead to resistance to TGF-ß. The model also allows the prediction of the integrated signalling pathway responses to the mutation of key components, e.g. Gp130 F/F.

Evolutionary suppression of erythropoiesis via the modulation of TGF-ß signalling in an Antarctic icefish.

The Antarctic icefish, a family (Channichthyidae) of teleosts within the perciform suborder Notothenioidei, are the only known vertebrates without oxygen-transporting haemoglobins and that are largely devoid of circulating erythrocytes. To elucidate the evo-devo mechanisms underpinning the suppressed erythropoiesis in the icefish, we conducted comparative studies on the transcriptomes and microRNAomes of the primary haematopoietic tissues between an icefish (Chionodraco hamatus) and two red-blooded notothenioids (Trematomus bernacchii and Gymnodraco acuticeps). We identified substantial remodelling of the haematopoietic programs in the icefish through which erythropoiesis is selectively suppressed. Experimental verification showed that erythropoietic suppression in the icefish may be attributable to the upregulation of TGF-ß signalling, which coincides with reductions in multiple transcription factors essential for erythropoiesis and the upregulation of hundreds of microRNAs, the majority (> 80%) of which potentially target erythropoiesis regulating factors. Of the six microRNAs selected for verification, three miRNAs (miR-152, miR-1388 and miR-16b) demonstrated suppressive functions on GATA1 and ALAS2, which are two factors important for erythroid differentiation, resulting in reduced numbers of erythroids in microinjected zebra fish embryos. Codon substitution analyses of the genes of the TGF-ß superfamily revealed signs of positive selection in TGF-ß1 and endoglin in the lineages leading to Antarctic notothenioids. Both genes are previously known to function in erythropoietic suppression. These findings implied a general trend of erythropoietic suppression in the cold-adapted notothenioid lineages through evolutionary modulation of the multi-functional TGF-ß signalling pathway. This trend is more pronounced in the haemoglobin-less icefish, which may pre-emptively hinder the otherwise defective erythroids from production.

Cancer-associated fibroblasts barrier breaking via TGF-ß blockade paved way for docetaxel micelles delivery to treat pancreatic cancer.

TGF-ß is a crucial regulator in tumor microenvironment (TME), especially for myofibroblastic cancer-associated fibroblasts (myCAFs). The myCAFs can be motivated by TGF-ß signaling to erect pro-tumor TME, meanwhile, myCAFs overexpress TGF-ß to mediate the crosstalk between tumor and stromal cells. The blockade of TGF-ß can break cancer-associated fibroblasts barrier, consequently opening the access for drugs into tumor. The TGF-ß is a promising target in anti-tumor therapy. Herein, we introduced a two-stage combination therapy (TC-Therapy), including TGF-ß receptor I inhibitor SB525334 (SB) and cytotoxicity agent docetaxel micelle (DTX-M). We found that SB and DTX-M synergistically inhibited myCAFs proliferation and elevated p53 protein expression in BxPC-3/3T3 mixed cells. Gene and protein tests demonstrated that SB cut off TGF-ß signaling via receptor blockade and it did not arouse TGF-ß legend compensated internal autocrine. On the contrary, two agents combined decreased TGF-ß secretion and inhibited myCAFs viability marked by α-SMA and FAPα. TC-Therapy was applied in BxPc-3/3T3 mixed tumor-bearing mice model. After TC-Therapy, the α-SMA+/ FAPα+ myCAFs faded increasingly and collagenous fibers mainly secreted by myCAFs decreased dramatically as well. More than that, the myCAFs barrier breaking helped to normalize micro-vessels and paved way for micelle penetration. The TGF-ß protein level of TC-Therapy in TME was much lower than that of simplex DTX-M, which might account for TME restoration. In conclusion, TGF-ß inhibitor acted as the pioneer before nano chemotherapeutic agents. The TC-Therapy of TGF-ß signaling inhibition and anti-tumor agent DTX-M is a promising regimen without arising metastasis risk to treat pancreatic cancer. The therapeutic regimen focused on TGF-ß related myCAFs reminds clinicians to have a comprehensive understanding of pancreatic cancer.