Canonical TGF-β Signaling Pathway Research Articles

Verteporfin inhibits TGF-β signaling by disrupting the Smad2/3-Smad4 interaction.

Transforming growth factor-β (TGF-β) signaling plays a crucial role in pathogenesis, such as accelerating tissue fibrosis and promoting tumor development at the later stages of tumorigenesis by promoting epithelial-mesenchymal transition, cancer cell migration, and invasion. Targeting TGF-β signaling is a promising therapeutic approach, but non-specific inhibition may result in adverse effects. In this study, we focus on the Smad2/3-Smad4 complex, a key component in TGF-β signaling transduction, as a potential target for cancer therapy. Through a phase-separated condensate-aided biomolecular interaction system, we identified verteporfin (VP) as a small-molecule inhibitor that specifically targets the Smad2/3-Smad4 interaction. VP effectively disrupted the interaction between Smad2/3 and Smad4 and thereby inhibited canonical TGF-β signaling, but not the interaction between Smad1 and Smad4 in BMP signaling. Furthermore, VP exhibited inhibitory effects on TGF-β-induced epithelial-mesenchymal transition and cell migration. Our findings indicate a novel approach to develop protein-protein interaction inhibitors of the canonical TGF-β signaling pathway for treatments of related diseases.

Complex and variable regulation of ΔNp63 and TAp63 by TGFβ has implications for the dynamics of squamous cell epithelial to mesenchymal transition

TGFβ has roles in inflammation, wound healing, epithelial to mesenchymal transition (EMT), and cancer stem cell states, and acts as a tumor suppressor gene for squamous cell carcinoma (SCC). SCCs are also characterized by high levels of ΔNp63, which induces epithelial cell phenotypes and maintains squamous stem cells. Previous studies indicate a complex interplay between ΔNp63 and TGFβ signaling, with contradictory effects reported. We investigated the effects of TGFβ on p63 isoform proteins and mRNAs in non-malignant squamous and SCC cells, and the role of either canonical or non-canonical TGFβ signaling pathways. TGFβ selectively increased ΔNp63 protein levels in non-malignant keratinocytes in association with SMAD3 activation and was prevented by TGFβ receptor inhibition, indicating activation of canonical TGFβ pathway signaling. TP63 isoform mRNAs showed discordance from protein levels, with an initial increase in both TAP63 and ΔNP63 mRNAs followed by a decrease at later times. These data demonstrate complex and heterogeneous effects of TGFβ in squamous cells that depend on the extent of canonical TGFβ pathway aberrations. The interplay between TGFβ and p63 is likely to influence the magnitude of EMT states in SCC, with clinical implications for tumor progression and response to therapy.

Copper chelation suppresses epithelial-mesenchymal transition by inhibition of canonical and non-canonical TGF-β signaling pathways in cancer

BackgroundMetastatic cancer cells exploit Epithelial-mesenchymal-transition (EMT) to enhance their migration, invasion, and resistance to treatments. Recent studies highlight that elevated levels of copper are implicated in cancer progression and metastasis. Clinical trials using copper chelators are associated with improved patient survival; however, the molecular mechanisms by which copper depletion inhibits tumor progression and metastasis are poorly understood. This remains a major hurdle to the clinical translation of copper chelators. Here, we propose that copper chelation inhibits metastasis by reducing TGF-β levels and EMT signaling. Given that many drugs targeting TGF-β have failed in clinical trials, partly because of severe side effects arising in patients, we hypothesized that copper chelation therapy might be a less toxic alternative to target the TGF-β/EMT axis.ResultsOur cytokine array and RNA-seq data suggested a link between copper homeostasis, TGF-β and EMT process. To validate this hypothesis, we performed single-cell imaging, protein assays, and in vivo studies. Here, we used the copper chelating agent TEPA to block copper trafficking. Our in vivo study showed a reduction of TGF-β levels and metastasis to the lung in the TNBC mouse model. Mechanistically, TEPA significantly downregulated canonical (TGF-β/SMAD2&3) and non-canonical (TGF-β/PI3K/AKT, TGF-β/RAS/RAF/MEK/ERK, and TGF-β/WNT/β-catenin) TGF-β signaling pathways. Additionally, EMT markers of MMP-9, MMP-14, Vimentin, β-catenin, ZEB1, and p-SMAD2 were downregulated, and EMT transcription factors of SNAI1, ZEB1, and p-SMAD2 accumulated in the cytoplasm after treatment.ConclusionsOur study suggests that copper chelation therapy represents a potentially effective therapeutic approach for targeting TGF-β and inhibiting EMT in a diverse range of cancers.

Tumor-Induced T Cell Polarization by Schwann Cells.

Nerve-cancer crosstalk resulting in either tumor neurogenesis or intratumoral neurodegeneration is critically controlled by Schwann cells, the principal glial cells of the peripheral nervous system. Though the direct stimulating effect of Schwann cells on malignant cell proliferation, motility, epithelial-mesenchymal transition, and the formation of metastases have been intensively investigated, the ability of Schwann cells to affect the effector and regulatory immune cells in the tumor environment is significantly less studied. Here, we demonstrated that tumor cells could stimulate Schwann cells to produce high levels of prostaglandin E, which could be blocked by COX-2 inhibitors. This effect was mediated by tumor-derived TGF-β as neutralization of this cytokine in the tumor-conditioned medium completely blocked the inducible prostaglandin E production by Schwann cells. Similar protective effects were also induced by the Schwann cell pretreatment with TGF-βR1/ALK4/5/7 and MAPK/ERK kinase inhibitors of the canonical and non-canonical TGF-β signaling pathways, respectively. Furthermore, prostaglandin E derived from tumor-activated Schwann cells blocked the proliferation of CD3/CD28-activated T cells and upregulated the expression of CD73 and PD-1 on both CD4+ and CD8+ T cells, suggesting T cell polarization to the exhausted phenotype. This new pathway of tumor-induced T cell inhibition via the activation of neuroglial cells represents new evidence of the importance of nerve-cancer crosstalk in controlling tumor development and progression. A better understanding of the tumor-neuro-immune axis supports the development of efficient targets for harnessing this axis and improving the efficacy of cancer therapy.

Tumor-produced and aging-associated oncometabolite methylmalonic acid promotes cancer-associated fibroblast activation to drive metastatic progression

The systemic metabolic shifts that occur during aging and the local metabolic alterations of a tumor, its stroma and their communication cooperate to establish a unique tumor microenvironment (TME) fostering cancer progression. Here, we show that methylmalonic acid (MMA), an aging-increased oncometabolite also produced by aggressive cancer cells, activates fibroblasts in the TME, which reciprocally secrete IL-6 loaded extracellular vesicles (EVs) that drive cancer progression, drug resistance and metastasis. The cancer-associated fibroblast (CAF)-released EV cargo is modified as a result of reactive oxygen species (ROS) generation and activation of the canonical and noncanonical TGFβ signaling pathways. EV-associated IL-6 functions as a stroma-tumor messenger, activating the JAK/STAT3 and TGFβ signaling pathways in tumor cells and promoting pro-aggressive behaviors. Our findings define the role of MMA in CAF activation to drive metastatic reprogramming, unveiling potential therapeutic avenues to target MMA at the nexus of aging, the tumor microenvironment and metastasis.

Antitumor Effect of Demethylzeylasteral (T-96) on Triple-Negative Breast Cancer via LSD1-Mediate Epigenetic Mechanisms.

Background and Purpose. Breast cancer ranks first in the incidence of female tumors. Triple-negative breast cancer (TNBC), one type of breast cancer, is more aggressive and has a worse prognosis. Demethylzeylasteral (T-96) is isolated from Tripterygium wilfordii Hook F. Our previous study found that T96 could inhibit TNBC invasion via suppressing the canonical and noncanonical TGF-β signaling pathways. However, the antitumor effects and mechanisms of T-96 on TNBC have not been studied. This study is aimed at investigating the antitumor effect and mechanism of T-96 on breast cancer. Experimental approach. MTT assay, Live and Dead cell assay, and TUNEL were used to observe the antitumor effect of breast cancer cells treated with T-96. siRNA of LSD1, Co-IP, and molecular docking were used to explore the direct target and mechanism of T-96. Subcutaneous murine xenograft models were used to detect the efficacy of T-96 antitumor activity in vivo. Key Results. T-96 was more susceptible to inducing the apoptosis of highly metastatic TNBC cell lines (SUM-1315). An abnormal level of histone methylation is a crucial characteristic of metastatic cancer cells. LSD1 is a histone demethylase. We found that T-96 could significantly decrease the protein expression of LSD1, increase its target protein PTEN expression and enhance histone methylation. T-96 could also down-regulate the PI3K/AKT signaling pathway, which could be blocked by PTEN. Knockdown of LSD1 by siRNA blocked the pharmacological activity of T-96. And the molecular docking predicted T-96 processed affinity toward LSD1 through hydrogen bonding. Finally, T-96 was evaluated in a murine xenograft model of SUM-1315 cells. And T-96 could significantly inhibit tumor growth without showing marked toxicity. Conclusions & Implications. The results illustrated that T-96 exerted antitumor activity in highly metastatic TNBC by inactivating the LSD1 function.

Apoptotic and Anti-metastatic Effects of Atractylodes lancea (Thunb.) DC. in a Hamster Model of Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a highly aggressive tumor with a greater risk of distant metastasis. The promising anti-CCA activity and safety profile of Atractylodes lancea (AL) have previously been reported in a series of in vitro, in vivo and clinical studies. The present study investigated the effect of AL extract on apoptosis and metastasis signaling pathways in the Opisthorchis viverrini/dimethylnitrosamine (OV/DMN)-induced CCA hamster model. Hamster liver tissues were obtained from the four groups (n = 5 per group), i.e., (i) 5-FU treated CCA (40 µg/mL); (ii) CCA; (iii) AL-treated CCA (5,000 mg/kg), and (iv) normal hamsters. Total RNA was isolated, and the expression levels of apoptosis-related and metastasis-related genes were determined by qRT-PCR analysis. The expression levels of p16, caspase-3, caspase-8, caspase-9, Apaf-1, p53 and Eef1a1 were downregulated, while that of the remaining genes were upregulated in CCA hamsters compared with normal hamsters. AL treatment increased the expression of p16, caspase-9, caspase-3, Apaf-1, p53 and E-cadherin and decreased the expression of cyclin D1, cdk4, Bax, Akt/PKB, Bcl-2, Mfge-8, Lass4, S100A6, TGF-β, Smad-2, Smad-3, Smad-4, MMP-9, and N-cadherin. The expression of Eef1a1 was unchanged. The anti-CCA activity of AL in OV/DMN-induced CCA hamsters could be due to the induction of cell cycle arrest at the G1 phase and activation of the apoptosis pathway, resulting in cancer cell death. The activation of the apoptosis pathway mainly involved the intrinsic pathway (activation of caspase-3 and caspase-9 through p53 and Mfge-8 modulation and downregulation of anti-apoptotic genes Akt and Bcl-2). In addition, AL could also inhibit the canonical TGF-β signaling pathway, MMP-9 and N-cadherin to suppress tumor metastasis.

Abstract 6230: Loss of ALK4 function promotes cancer progression by regulating cell surface TGF-β receptor stability

Abstract Pancreatic cancer is a lethal disease. More than 50% of pancreatic cancer patients are diagnosed with metastatic disease with a dismal 3% five-year survival rate. There is clearly an urgent need to identify novel targets to treat pancreatic cancer patients. Transforming growth factor-β (TGF-β) signaling is dysregulated during cancer progression, inhibiting tumorigenesis while promoting metastasis. Approximately half of pancreatic cancer patients have mutations of genes in the TGF-β signaling pathway, including SMAD4, SMAD3, TGFBR1, TFGBR2, ACVR1B, and ACVR2A. ALK4 (Activin receptor-like kinase 4), which is encoded by ACVR1B, is a type I receptor in the TGF-β superfamily. Mutation or copy number loss of ALK4 occurs in 34% of pancreatic cancer patients, while ALK4 expression is significantly down-regulated in many cancers, including pancreatic cancer, with low ALK4 expression associated with poor clinical outcomes. Here we demonstrate that loss of ALK4 increases cell migration and invasion, induces epithelial to mesenchymal transition (EMT), and promotes local and distal metastasis in pancreatic cancer models. Loss of ALK4 unexpectedly increases activation of the canonical TGF-β signaling pathway and significantly induces TGF-β-mediated expression of EMT mediators and effectors. Moreover, ALK4 loss increases the cell surface expression of TβRs (TGFβ receptor type I and II), without changing the receptor gene expression, by increasing N-linked glycosylation of TβRs, potentially by altering Golgi morphology and function. Proteomic analysis of newly synthesized proteins induced by ALK4 loss identified galectin-3 (Gal-3) as upregulated. Gal-3 is known to be upregulated by MGAT5 (Alpha-1,6-Mannosylglycoprotein 6-Beta-N-Acetylglucosaminyltransferase) and cross-links MGAT5-modified N-glycans on TβRs at the cell surface, suppressing TβRs endocytosis. Consistent with this observation, loss of ALK4 increased MGAT5 mRNA expression and induced Gal-3 protein expression, suggesting that ALK4 might regulate TβRs stability by promoting lattice formation with Gal-3 and preventing internalization. Taken together, our results suggest that the loss of ALK4 increases TβR receptor stability through regulating receptor glycosylation, activating TGF-β signaling, promoting the EMT transition and cancer progression. Citation Format: Manqi Zhang, Jian Chen, Gerard Conrad Blobe. Loss of ALK4 function promotes cancer progression by regulating cell surface TGF-β receptor stability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6230.

Asthmatic Eosinophils Alter the Gene Expression of Extracellular Matrix Proteins in Airway Smooth Muscle Cells and Pulmonary Fibroblasts.

The impaired production of extracellular matrix (ECM) proteins by airway smooth muscle cells (ASMC) and pulmonary fibroblasts (PF) is a part of airway remodeling in asthma. This process might be influenced by eosinophils that migrate to the airway and abundantly secrete various cytokines, including TGF-β. We aimed to investigate the effect of asthmatic eosinophils on the gene expression of ECM proteins in ASMC and PF. A total of 34 study subjects were recruited: 14 with allergic asthma (AA), 9 with severe non-allergic eosinophilic asthma (SNEA), and 11 healthy subjects (HS). All AA patients underwent bronchial allergen challenge with D. pteronyssinus. The peripheral blood eosinophils were isolated using high-density centrifugation and magnetic separation. The individual cell cultures were made using hTERT ASMC and MRC-5 cell lines and the subjects’ eosinophils. The gene expression of ECM and the TGF-β signaling pathway was analyzed using qRT-PCR. We found that asthmatic eosinophils significantly promoted collagen I, fibronectin, versican, tenascin C, decorin, vitronectin, periostin, vimentin, MMP-9, ADAM33, TIMP-1, and TIMP-2 gene expression in ASMC and collagen I, collagen III, fibronectin, elastin, decorin, MMP-2, and TIMP-2 gene expression in PF compared with the HS eosinophil effect. The asthmatic eosinophils significantly increased the gene expression of several canonical and non-canonical TGF-β signaling pathway components in ASMC and PF compared with the HS eosinophil effect. The allergen-activated AA and SNEA eosinophils had a greater effect on these changes. In conclusion, asthmatic eosinophils, especially SNEA and allergen-activated eosinophils, imbalanced the gene expression of ECM proteins and their degradation-regulating proteins. These changes were associated with increased gene expression of TGF-β signaling pathway molecules in ASMC and PF.

Expanding the mutation and phenotype spectrum of MYH3-associated skeletal disorders

Pathogenic variants in MYH3 cause distal arthrogryposis type 2A and type 2B3 as well as contractures, pterygia and spondylocarpotarsal fusion syndromes types 1A and 1B. These disorders are ultra-rare and their natural course and phenotypic variability are not well described. In this study, we summarize the clinical features and genetic findings of 17 patients from 10 unrelated families with vertebral malformations caused by dominant or recessive pathogenic variants in MYH3. Twelve novel pathogenic variants in MYH3 (NM_002470.4) were identified: three of them were de novo or inherited in autosomal dominant way and nine were inherited in autosomal recessive way. The patients had vertebral segmentation anomalies accompanied with variable joint contractures, short stature and dysmorphic facial features. There was a significant phenotypic overlap between dominant and recessive MYH3-associated conditions regarding the degree of short stature as well as the number of vertebral fusions. All monoallelic variants caused significantly decreased SMAD3 phosphorylation, which is consistent with the previously proposed pathogenic mechanism of impaired canonical TGF-β signaling. Most of the biallelic variants were predicted to be protein-truncating, while one missense variant c.4244T>G,p.(Leu1415Arg), which was inherited in an autosomal recessive way, was found to alter the phosphorylation level of p38, suggesting an inhibition of the non-canonical pathway of TGF-β signaling. In conclusion, the identification of 12 novel pathogenic variants and overlapping phenotypes in 17 affected individuals from 10 unrelated families expands the mutation and phenotype spectrum of MYH3-associated skeletal disorders. We show that disturbances of canonical or non-canonical TGF-β signaling pathways are involved in pathogenesis of MYH3-associated skeletal fusion (MASF) syndrome.

TGF-β and Cancer Immunotherapy.

The cytokine, transforming growth factor beta (TGF-β), has a history of more than 40 years. TGF-β is secreted by many tumor cells and is associated with tumor growth and cancer immunity. The canonical TGF-β signaling pathway, SMAD, controls both tumor metastasis and immune regulation, thereby regulating cancer immunity. TGF-β regulates multiple types of immune cells in tumor microenvironment, including T cells, natural killer (NK) cells, and macrophages. One of the main roles of TGF-β in the tumor microenvironment is the generation of regulatory T cells, which contribute to the suppression of anti-tumor immunity. Because cancer is one of the highest causes of death globally, the discovery of immune checkpoint inhibitors by Honjo and Allison in cancer immunotherapy earned a Nobel Prize in 2018. TGF-β also regulates the levels of immune checkpoints inhibitory receptors on immune cells. Immune checkpoints inhibitors are now being developed along with anti-TGF-β antibody and/or TGF-β inhibitors. More recently, chimeric antigen receptors (CARs) were applied to cancer immunity and tried to combine with TGF-β blockers.

A linkage between effectual genes in progression of CRC through canonical and non-canonical TGF-β signaling pathways.

Different molecular signaling pathways have been involved in the incidence and progression of CRC. We aimed to examine the correlation between eight candidate genes, including TFGβ, SMAD2, SMAD4, RhoA, EGFR, MAP2K1, MTA1, and LEF1 in the progression of colorectal cancer (CRC) and their association with clinicopathological variables and CRC patients prognosis. Immunohistochemistry and quantitative real-time polymerase chain reaction (qRT-PCR) analysis 2-ΔΔct, were performed to assess the expression of eight genes in 64 and 122 patients with CRC, respectively and 20 normal samples were added for verification. We showed a positive correlation between SMAD2 and MAP2K1 (r = 0.337, P < 0.001), MAP2K1 and LEF1 (r = 0.187, P = 0.03), SMAD4 and RhoA (r = 0.214, P = 0.01) and as well, a negative correlation between SMAD2 and TGFβ (r = - 0.197, P = 0.02), and RhoA and LEF1 (r = - 0.180, P = 0.04) in tumor tissues. A decrease in RhoA mRNA expression was associated with the advanced TNM stage (P = 0.01), while the EGFR and SMAD2 mRNA expression upregulated in advanced stages (P = 0.03, P = 0.03), respectively. Also, an increase in EGFR and SMAD4 protein expression was significantly associated with the advanced TNM stage (P = 0.000) (P = .002), respectively. Perceiving the connections between canonical and non-canonical Transforming growth factor (TGF-β) signaling pathway along with the epidermal growth factor receptor (EGFR) and WNT cascades may trigger the development of novel approaches for CRC prediction.

Phosphoinositide-binding activity of Smad2 is essential for its function in TGF-β signaling

As a central player in the canonical TGF-β signaling pathway, Smad2 transmits the activation of TGF-β receptors at the plasma membrane (PM) to transcriptional regulation in the nucleus. Although it has been well established that binding of TGF-β to its receptors leads to the recruitment and activation of Smad2, the spatiotemporal mechanism by which Smad2 is recruited to the activated TGF-β receptor complex and activated is not fully understood. Here we show that Smad2 selectively and tightly binds phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) in the PM. The PI(4,5)P2-binding site is located in the MH2 domain that is involved in interaction with the TGF-β receptor I that transduces TGF-β-receptor binding to downstream signaling proteins. Quantitative optical imaging analyses show that PM recruitment of Smad2 is triggered by its interaction with PI(4,5)P2 that is locally enriched near the activated TGF-β receptor complex, leading to its binding to the TGF-β receptor I. The PI(4,5)P2-binding activity of Smad2 is essential for the TGF-β-stimulated phosphorylation, nuclear transport, and transcriptional activity of Smad2. Structural comparison of all Smad MH2 domains suggests that membrane lipids may also interact with other Smad proteins and regulate their function in diverse TGF-β-mediated biological processes.

β-cell Smad2 null mice have improved β-cell function and are protected from diet-induced hyperglycemia

Understanding signaling pathways that regulate pancreatic β-cell function to produce, store, and release insulin, as well as pathways that control β-cell proliferation, is vital to find new treatments for diabetes mellitus. Transforming growth factor-beta (TGF-β) signaling is involved in a broad range of β-cell functions. The canonical TGF-β signaling pathway functions through intracellular smads, including smad2 and smad3, to regulate cell development, proliferation, differentiation, and function in many organs. Here, we demonstrate the role of TGF-β/smad2 signaling in regulating mature β-cell proliferation and function using β-cell-specific smad2 null mutant mice. β-cell-specific smad2-deficient mice exhibited improved glucose clearance as demonstrated by glucose tolerance testing, enhanced in vivo and ex vivo glucose-stimulated insulin secretion, and increased β-cell mass and proliferation. Furthermore, when these mice were fed a high-fat diet to induce hyperglycemia, they again showed improved glucose tolerance, insulin secretion, and insulin sensitivity. In addition, ex vivo analysis of smad2-deficient islets showed that they displayed increased glucose-stimulated insulin secretion and upregulation of genes involved in insulin synthesis and insulin secretion. Thus, we conclude that smad2 could represent an attractive therapeutic target for type 2 diabetes mellitus.

SMAD4 Feedback Activates the Canonical TGF-β Family Signaling Pathways.

TGF-β family signaling pathways, including TGF-β and BMP pathways, are widely involved in the regulation of health and diseases through downstream SMADs, which are also regulated by multiple validated mechanisms, such as genetic regulation, epigenetic regulation, and feedback regulation. However, it is still unclear whether R-SMADs or Co-SMAD can feedback regulate the TGF-β family signaling pathways in granulosa cells (GCs). In this study, we report a novel mechanism underlying the feedback regulation of TGF-β family signaling pathways, i.e., SMAD4, the only Co-SMAD, positive feedback activates the TGF-β family signaling pathways in GCs with a basal level of TGF-β ligands by interacting with the core promoters of its upstream receptors. Mechanistically, SMAD4 acts as a transcription factor, and feedback activates the transcription of its upstream receptors, including ACVR1B, BMPR2, and TGFBR2, of the canonical TGF-β signaling pathways by interacting with three coactivators (c-JUN, CREB1, and SP1), respectively. Notably, three different interaction modes between SMAD4 and coactivators were identified in SMAD4-mediated feedback regulation of upstream receptors through reciprocal ChIP assays. Our findings in the present study indicate for the first time that SMAD4 feedback activates the canonical TGF-β family signaling pathways in GCs, which improves and expands the regulatory mechanism, especially the feedback regulation modes of TGF-β family signaling pathways in ovarian GCs.

MicroRNAs in Transforming Growth Factor-Beta Signaling Pathway Associated With Fibrosis Involving Different Systems of the Human Body.

Fibrosis, a major cause of morbidity and mortality, is a histopathological manifestation of many chronic inflammatory diseases affecting different systems of the human body. Two types of transforming growth factor beta (TGF-β) signaling pathways regulate fibrosis: the canonical TGF-β signaling pathway, represented by SMAD-2 and SMAD-3, and the noncanonical pathway, which functions without SMAD-2/3 participation and currently includes TGF-β/mitogen-activated protein kinases, TGF-β/SMAD-1/5, TGF-β/phosphatidylinositol-3-kinase/Akt, TGF-β/Janus kinase/signal transducer and activator of transcription protein-3, and TGF-β/rho-associated coiled-coil containing kinase signaling pathways. MicroRNA (miRNA), a type of non-coding single-stranded small RNA, comprises approximately 22 nucleotides encoded by endogenous genes, which can regulate physiological and pathological processes in fibrotic diseases, particularly affecting organs such as the liver, the kidney, the lungs, and the heart. The aim of this review is to introduce the characteristics of the canonical and non-canonical TGF-β signaling pathways and to classify miRNAs with regulatory effects on these two pathways based on the influenced organ. Further, we aim to summarize the limitations of the current research of the mechanisms of fibrosis, provide insights into possible future research directions, and propose therapeutic options for fibrosis.

Determining the function of LvSmad3 on Litopenaeus vannamei in response to acute low temperature stress

Smad3 is a key mediator of the canonical TGF-β signaling pathway and plays an important role in TGF-β1-mediated transcriptional regulation. However, the function of Smad3 in crustaceans such as shrimp, is still poorly understood and needs to be further explored. We characterized Litopenaeus vannamei Smad3 (LvSmad3) and its biological functions were investigated in response low temperature stress. Full-length LvSmad3 cDNA was 2341bp and contained an open reading frame (ORF) of 1326 bp that encoded a 441 amino acid long protein, with a predicted molecular mass of 48.35 kDa. Phylogenetic analysis revealed that LvSmad3 has a high degree of similarity with other known species. LvSmad3 mRNA was detected in all the tested tissues and highest transcription occurred mostly in gills. Further research showed that suppressing the expression of Smad3 could reduce ROS production, DNA damage and the apoptosis rate in shrimp hemocyte under low temperature compared with the dsGFP group. Thus, we speculated that Smad3 could promote the apoptosis of hemocytes. We confirmed that Smad3 could inhibit apoptosis in the hepatopancreas by suppressing the expression of pro-apoptotic genes. Taken together, the silencing of Smad3 can reduce ROS production induced by low temperature stress, weaken the damage to hemocytes and the hepatopancreas by inhibit the apoptosis.

Chordacentrum mineralization is delayed in zebrafish betaglycan-null mutants.

The Transforming Growth Factor β (TGFβ) family is a group of related proteins that signal through a type I and type II receptors. Betaglycan, also known as the type III receptor (Tgfbr3), is a coreceptor for various ligands of the TGFβ family that participates in heart, liver and kidney development as revealed by the tgfbr3-null mouse, as well as in angiogenesis as revealed by Tgfbr3 downregulation in morphant zebrafish. Here, we present CRISPR/Cas9-derived zebrafish Tgfbr3-null mutants, which exhibited unaltered embryonic angiogenesis and developed into fertile adults. One reproducible phenotype displayed by these Tgfbr3-null mutants is delayed chordacentra mineralization, which nonetheless does not result in vertebral abnormalities in the adult fishes. We also report that the canonical TGFβ signaling pathway is needed for proper chordacentra mineralization and that Tgfbr3 absence decreases this signal in the notochordal cells responsible for this process. Betaglycan's "ligand presentation" function contributes to the optimal TGFβ signaling required for zebrafish chordacentra mineralization.

Effects of emodin, a plant-derived anthraquinone, on TGF-β1-induced cardiac fibroblast activation and function.

Cardiac fibrosis accompanies a number of pathological conditions and results in altered myocardial structure, biomechanical properties and function. The signaling networks leading to fibrosis are complex, contributing to the general lack of progress in identifying effective therapeutic approaches to prevent or reverse this condition. Several studies have shown protective effects of emodin, a plant-derived anthraquinone, in animal models of fibrosis. A number of questions remain regarding the mechanisms whereby emodin impacts fibrosis. Transforming growth factor beta 1 (TGF-β1) is a potent stimulus of fibrosis and fibroblast activation. In the present study, experiments were performed to evaluate the effects of emodin on activation and function of cardiac fibroblasts following treatment with TGF-β1. We demonstrate that emodin attenuates TGF-β1-induced fibroblast activation and collagen accumulation in vitro. Emodin also inhibits activation of several canonical (SMAD2/3) and noncanonical (Erk1/2) TGF-β signaling pathways, while activating the p38 pathway. These results suggest that emodin may provide an effective therapeutic agent for fibrosis that functions via specific TGF-β signaling pathways.

Physical Training Inhibits the Fibrosis Formation in Alzheimer’s Disease Kidney Influencing the TGFβ Signaling Pathways

Background:Alzheimer’s disease (AD) is a neurodegenerative illness, with several peripheral pathological signs such as accumulation of amyloid-β (Aβ) plaques in the kidney. Alterations of transforming growth factor β (TGFβ) signaling in the kidney can induce fibrosis, thus disturbing the elimination of Aβ.Objective:A protective role of increased physical activity has been proven in AD and in kidney fibrosis, but it is not clear whether TGFβ signalization is involved in this effect.Methods:The effects of long-term training on fibrosis were investigated in the kidneys of mice representing a model of AD (B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J) by comparing wild type and AD organs. Alterations of canonical and non-canonical TGFβ signaling pathways were followed with PCR, western blot, and immunohistochemistry.Results:Accumulation of collagen type I and interstitial fibrosis were reduced in kidneys of AD mice after long-term training. AD induced the activation of canonical and non-canonical TGFβ pathways in non-trained mice, while expression levels of signal molecules of both TGFβ pathways became normalized in trained AD mice. Decreased amounts of phosphoproteins with molecular weight corresponding to that of tau and the cleaved C-terminal of AβPP were detected upon exercising, along with a significant increase of PP2A catalytic subunit expression.Conclusion:Our data suggest that physical training has beneficial effects on fibrosis formation in kidneys of AD mice and TGFβ signaling plays a role in this phenomenon.