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TGF-β-SMAD signaling pathway plays an important role in the progression of various cancers. However, posttranscriptional regulation such as N 6 -methyladenosine (m 6 A) of TGF-β-SMAD signaling axis remains incompletely understood. Here, we reveal that insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2) is low expression as well as associated with poor prognosis in clear cell renal cell carcinoma (ccRCC) patients and inhibits proliferation as well as promotes metastasis of ccRCC cells. Mechanistically, IGF2BP2 systematically regulates TGF-β-SMAD signaling family, including TGF-β1/2, TGF-βR1/2 and SMAD2/3/4, through mediating their mRNA stability in an m 6 A-dependent manner. Furthermore, the functional effects of IGF2BP2 on ccRCC cells is mediated by TGF-β-SMAD signaling downstream effector SMAD4, which is identified three m 6 A sites in 5'UTR and CDS. Our study establishes IGF2BP2-TGF-β-SMAD axis as a new regulatory effector in ccRCC, providing new insights for developing novel therapeutic strategies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The aim of this study was to determine the osteogenic activity and mechanism of soybean peptide VVELLKAFEEKF (SOP) and the potential relationship between SOP and transforming growth factor-β1 (TGF-β1). The results show that SOP promotes MC3T3-E1 cell proliferation by altering cell progression. SOP induced cell differentiation and mineralization in a dose-dependent manner at 0.7-7 μM. Moreover, SOP stimulates osteoblast differentiation, which may be achieved through the activation of p38-MAPK and Smad2/3 signaling pathways. Furthermore, treatment with a TβRI inhibitor (SB525334) inhibited the phosphorylation levels of p38 and Smad2/3, which indicates the involvement of TβRI in the process of osteoblast differentiation caused by SOP. Besides, in non-FBS-cultured MC3T3-E1 cells, SOP and TGF-β1 promoted the phosphorylation of Smad2/3 and alkaline phosphatase (ALP) activity, but the effect was lost when SOP was incubated separately, indicating that SOP stimulated osteoblast differentiation by promoting TGF-β1 activity. In vivo , SOP significantly restores bone mineral density loss and behavioral deficits in a model of glucocorticoid-induced osteoporosis (GIOP) in zebrafish. These results suggest that SOP may have the function of promoting bone remodeling and may be used as a potential active factor for functional food development to prevent osteoporosis.

Background: Fatty liver disease is a metabolic disorder that recently has been classified into two categories: metabolic dysfunction-associated fatty liver disease (MAFLD) and non-MAFLD. TGF-β signaling pathway is likely a significant factor in the pathogenesis of this condition, exerting its effects through its downstream signaling proteins, Smad2/3. Accordingly, this study aimed to investigate the TGF-β signaling pathway in the white blood cells (WBCs) of patients with MAFLD compared to those with non-MAFLD and control groups., Methods and Results: In this study, 41 patients with fatty liver were evaluated, comprising 22 patients with MAFLD and 19 patients with non-MAFLD, and compared to 22 healthy controls. Gene expression of TGF-β1, TGF-β3, and CTGF were quantified using qRT-PCR, and the protein expressions of Smad2/3 and P-Smad2/3 were analyzed using western blotting. Gene expression analysis revealed a significant decrease in the gene expressions of the TGF-β1 and TGF-β3 and an increase in CTGF gene expression in patients with MAFLD and non-MAFLD compared to the control group. Notably, the Smad2/3 protein expression was significantly higher in the non-MAFLD group compared to the control group (P < 0.05). On the other hand, the P-smad2/3 protein expression was significantly elevated in the MAFLD group compared to the control group (P < 0.001)., Conclusions: TGF-β signaling pathway in WBCs of patients with fatty liver are affected by a complex signaling pathway. However, metabolic factors most probably affect TGF-β1 gene expression and its downstream signaling proteins more than TGF-β3., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The prevalence of chronic kidney disease (CKD) in dogs increases with age, and renal fibrosis is an important pathophysiological mechanism in this process. However, only a few drugs that can effectively inhibit fibrosis in the kidneys of dogs are currently available. In this study, we aimed to determine whether pirfenidone, a drug that has shown antifibrotic effects in various clinical studies, also exerts antifibrotic effects on canine renal tubular epithelial cells, Madin-Darby canine kidney cells (MDCK). To this end, we treated MDCK cells with various concentrations of pirfenidone, followed by transforming growth factor-beta1 (TGF-β1) to stimulate fibrotic conditions. A cell viability assay was performed to determine the effect of pirfenidone on cell survival. Fibrosis-related markers and TGF-β1 fibrotic pathway-related markers were assessed using qPCR, Western blot analysis and immunocytochemistry. A one-way analysis of variance (ANOVA) was performed, followed by Tukey's post-hoc test for multiple comparisons. Pirfenidone treatment significantly reduced the expression of profibrotic markers such as α-smooth muscle actin, fibronectin, and collagen. Additionally, it upregulated the expression of E-cadherin, an epithelial marker. Furthermore, pirfenidone effectively inhibited the phosphorylation of key factors involved in the TGF-β1 signaling pathway, including Smad2/3 and ERK1/2. These results demonstrate that pirfenidone suppresses TGF-β1-induced fibrosis in MDCK cells by attenuating epithelial-mesenchymal transition and the relevant signaling pathways., (© 2024. The Author(s).)

Purpose: This study investigated the effect of an isocitrate dehydrogenase 1 (IDH1) mutation (mutIDH1) on the invasion and angiogenesis of human glioma cells., Methods: Doxycycline was used to induce the expression of mutIDH1 in glioma cells. Transwell and wound healing assays were conducted to assess glioma cell migration and invasion. Western blotting and cell immunofluorescence were used to measure the expression levels of various proteins. The influence of bone morphogenetic protein 2 (BMP2) on invasion, angiogenesis-related factors, BMP2-related receptor expression, and changes in Smad signaling pathway-related proteins were evaluated after treatment with BMP2. Differential gene expression and reference transcription analysis were performed., Results: Successful infection with recombinant lentivirus expressing mutIDH1 was demonstrated. The IDH1 mutation promoted glioma cell migration and invasion while positively regulating the expression of vascularization-related factors and BMP2-related receptors. BMP2 exhibited a positive regulatory effect on the migration, invasion, and angiogenesis of mutIDH1-glioma cells, possibly mediated by BMP2-induced alterations in Smad signaling pathway-related factors.After BMP2 treatment, the differential genes of MutIDH1-glioma cells are closely related to the regulation of cell migration and cell adhesion, especially the regulation of Smad-related proteins. KEGG analysis confirmed that it was related to BMP signaling pathway and TGF-β signaling pathway and cell adhesion. Enrichment analysis of gene ontology and genome encyclopedia further confirmed the correlation of these pathways., Conclusion: Mutation of isocitrate dehydrogenase 1 promotes the migration, invasion, and angiogenesis of glioma cells, through its effects on the BMP2-driven Smad signaling pathway. In addition, BMP2 altered the transcriptional patterns of mutIDH1 glioma cells, enriching different gene loci in pathways associated with invasion, migration, and angiogenesis., (© 2024. The Author(s).)

The aim of this study was to identify miRNAs that could potentially influence the activity of SMAD proteins involved in TGFβ signal transduction in five types of breast cancer in Polish women. Patients with five breast cancer subtypes were included in the study: luminal A ( n = 130), luminal B HER2- ( n = 100), luminal B HER2+ ( n = 96), non-luminal HER2+ ( n = 36), and TNBC ( n = 43). During surgery, tumor tissue was removed along with a margin of healthy tissue (control). Molecular analysis included determination of the expression of genes related to SMAD protein signal transduction using mRNA microarrays and reverse transcription quantitative polymerase chain reaction (RT-qPCR). Protein expression was determined using an enzyme-linked immunosorbent assay (ELISA). The miRNA profiling was performed using miRNA microarrays and the miRDB database. SMAD3 and SMAD5 were overexpressed in all types of breast cancer, which could be related to the reduced expression of miR-145, and the findings for SMAD4 and miR-155 were similar. Additionally, the level of SMAD7 was reduced, which may be due to the low activity of miR-15b and miR21b. This study determined the gene expression profiles involved in SMAD protein signal transduction across five different types of breast cancer and identified the miRNAs potentially regulating their activity. Overexpression of SMAD3, SMAD4, and SMAD5 suggests excessive activation of the TGFβ pathway, potentially promoting tumor growth and development. Concurrently, a significant reduction in SMAD7 expression removes inhibitory control in the TGFβ pathway, a phenomenon that is particularly evident in more aggressive breast cancer types.

The primary cause of mortality in colorectal cancer (CRC) patients is tumor metastasis. The epithelial-mesenchymal transition (EMT) stands out as a crucial factor promoting the metastasis of CRC. Previous findings suggest a potential inhibitory effect of docosahexaenoic acid (DHA) on CRC metastasis, but the precise mechanism remains unknown, this study aims to explore this issue. We assessed metastasis and recurrence, all-cause mortality, and cancer-related mortality rates according to DHA intake in independent CRC cohorts ( n = 367) by survival analysis. The ability of DHA to block CRC cell migration and invasion was tested using transwell and wound-healing assays. The regulation of EMT marker genes in CRC by DHA was detected by quantitative real-time PCR (qPCR) and immunoblotting, and the effect of DHA on the TGF-β1/Smad signaling pathway was further investigated. These cellular findings were validated using a subcutaneous CRC mouse model. Survival analyses showed that lower DHA intake was associated with a higher risk of CRC metastasis and a poorer prognosis. In vitro experiments showed that DHA inhibits the TGF-β1/Smad signaling pathway and regulates downstream transcription factors, thereby reversing the EMT and inhibiting invasion and migration. In the mouse model, dietary DHA supplementation effectively increased blood DHA concentrations and inhibited CRC metastasis. Our study demonstrated that DHA inhibits CRC invasion and metastasis by inhibiting the TGF-β1/Smad signaling pathway. Increased intake of DHA among CRC patients may provide additional benefits to the prognosis of colorectal cancer.

Breast cancer is the second primary cause of cancer death among women. Long non-coding RNA (lncRNA) X-inactive specific transcript (XIST) is a central regulator for X chromosome inactivation, and its abnormal expression is a primary feature of breast cancer. So far, the mechanism of XIST in breast cancer has not been fully elucidated. We attempted to illustrate the mechanism of XIST in breast cancer. The expressions of XIST, microRNA-455-3p (miR-455-3p) in breast cancer were measured using quantitative real-time PCR. The expressions of homeobox C4 (HOXC4) were assessed with immunohistochemical and Western blot. Also, the functions of XIST in breast cancer were assessed by Cell Counting Kit-8 analysis, colony formation assay, flow cytometry, Western blot, Transwell, and cell scratch assays. Meanwhile, the mechanism of XIST in breast cancer was validated using database analysis and dual-luciferase reporter assay. Furthermore, the function of XIST in breast cancer in vivo was estimated by tumor xenograft model, immunohistochemical assay, and hematoxylin-eosin staining. XIST and HOXC4 expressions were increased, but miR-455-3p expressions were decreased in breast cancer tissues and cells. Knocking down XIST restrained breast cancer cell proliferation, invasion, migration, epithelial-mesenchymal transformation (EMT), and induced cell cycle arrest at G0/G1. Meanwhile, XIST interacted with miR-455-3p, while miR-455-3p interacted with HOXC4. XIST knockdown repressed breast cancer cell proliferation, invasion, and EMT, while miR-455-3p inhibitor or HOXC4 overexpression abolished those impacts. HOXC4 overexpression also blocked the impacts of miR-455-3p mimic on breast cancer cell malignant behavior. In vivo experimental data further indicated that XIST knockdown repressed breast cancer cell tumorigenic ability, and decreased HOXC4 and p-SMAD3 (TGF-β/SMAD-related protein) expressions.XIST/miR-455-3p/HOXC4 facilitated breast cancer development by activating the TGF-β/SMAD pathway., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Proton pump inhibitors (PPIs) are one of the most commonly prescribed medications. However, PPI usage is linked to a higher risk of both acute and chronic renal damage by mechanisms not entirely known. The present study demonstrates that omeprazole (10 mg/kg body weight, i.p.) causes TGF-β/Smad signaling activation and subsequent expression of the profibrotic genes CTGF and TIMP-1 in rat kidney. Increased production of CTGF and TIMP-1 accompany activation of the TGF-β/Smad signaling cascade. However, simultaneous treatment of omeprazole and the TGF-β inhibitor, disitertide (P144) (1 mg/kg body weight i.p.) suppresses the TGF-β/Smad signaling pathway and subsequent production of CTGF and TIMP-1. Additionally, TGF-β level in rat kidney was highly reduced in animals treated with the ROS (reactive oxygen species) scavenger, N-acetyl cysteine (NAC) (100 mg/kg body weight i.p.) before omeprazole administration. Furthermore, the reduction in SOD activity brought by omeprazole was returned to the normal level in those animals. However, MDA level increased by omeprazole was highly reduced in the presence of NAC. Collectively, the current findings demonstrate that omeprazole has the ability to promote the expression of the profibrotic genes CTGF and TIMP-1 in a ROS and TGF-β dependent manner. The present study suggests the co-use of ROS scavenger to improve the therapeutic use of the PPI omeprazole.

Background: Idiopathic pulmonary fibrosis (IPF) represents a severe and progressive manifestation of idiopathic interstitial pneumonia marked by an uncertain etiology along with an unfavorable prognosis. Osteoglycin (OGN), belonging to the small leucine-rich proteoglycans family, assumes pivotal functions in both tissue formation and damage response. However, the roles and potential mechanisms of OGN in the context of lung fibrosis remain unexplored., Methods: The assessment of OGN expression levels in fibrotic lungs was conducted across various experimental lung fibrosis mouse models. To elucidate the effects of OGN on the differentiation of lung myofibroblasts, both OGN knockdown and OGN overexpression were employed in vitro. The expression of integrin αv, along with its colocalization with lysosomes and latency-associated peptide (LAP), was monitored in OGN-knockdown lung myofibroblasts. Furthermore, the role of OGN in lung fibrosis was investigated through OGN knockdown utilizing adeno-related virus serotype 6 (AAV6)-mediated delivery., Results: OGN exhibited upregulation in both lungs and myofibroblasts across diverse lung fibrosis mouse models. And laboratory experiments in vitro demonstrated that OGN knockdown inhibited the TGF-β/Smad signaling pathway in lung myofibroblasts. Conversely, OGN overexpression promoted TGF-β/Smad pathway in these cells. Mechanistic insights revealed that OGN knockdown facilitated lysosome-mediated degradation of integrin αv while inhibiting its binding to latency-associated peptide (LAP). Remarkably, AAV6-targeted OGN knockdown ameliorated the extent of lung fibrosis in experimental mouse models., Conclusion: Our results indicate that inhibiting OGN signaling could serve as a promising therapeutic way for lung fibrosis., Competing Interests: Declaration of competing interest No conflicts of interest., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Here, we examined the effects of the BMP signaling pathway inhibitor LDN-193189 on the pluripotency of porcine embryonic stem cells (ESCs) in the absence of feeder cells using molecular and transcriptomic techniques. Additionally, the effects of some extracellular matrix components on porcine ESC pluripotency were evaluated to develop an optimized and sustainable feeder-free culture system for porcine ESCs. Feeder cells were found to play an important role in supporting the pluripotency of porcine ESCs by blocking trophoblast and mesodermal differentiation through the inhibition of the BMP pathway. Additionally, treatment with LDN-193189, an inhibitor of the BMP pathway, maintained the pluripotency and homogeneity of porcine ESCs for an extended period in the absence of feeder cells by stimulating the secretion of chemokines and suppressing differentiation, based on transcriptome analysis. Conclusively, these results suggest that LDN-193189 could be a suitable replacement for feeder cells in the maintenance of porcine ESC pluripotency during culture. Additionally, these findings contribute to the understanding of pluripotency gene networks and comparative embryogenesis., Competing Interests: Declaration of competing interest Authors declare that they have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

This study aimed to determine the function of angiopoietin-related protein 4 (ANGPTL4) and bone morphogenetic protein 7 (BMP7) on hepatocellular carcinoma (HCC). Overexpressing plasmids were cotransfected into HepG2 cells to determine the interaction between ANGPTL4 and BMP7. The effect of ANGPTL4 on the stability of BMP7 is examined by detecting the expression and ubiquitination levels. In vitro and in vivo experiments of knocking down ANGPTL4 while overexpressing BMP7 were performed to investigate whether the effects of ANGPTL4 on HCC proliferation, migration, and downstream signaling pathways were dependent on BMP7. ANGPTL4 is able to interact with BMP7, and knockdown of ANGPTL4 increased BMP7 expression and ubiquitination. Overexpression of BMP7 reversed the inhibition of HCC proliferation and migration as well as the decrease in the expression levels of Smad1/5/8 and MAPK14 caused by knockdown of ANGPTL4. ANGPTL4 promotes the proliferation and migration of HCC by inhibiting the ubiquitination degradation of BMP7 and the Smad/MAPK pathway, providing a novel mechanism and a potential therapeutic target for the treatment of HCC.

To investigate the effects of Luhong Yixin Granules on myocardial fibrosis in rats with heart failure and its possible mechanism, a total of 60 male Wistar rats were randomly divided into the control group, model group, and low-, medium-and high-dose Luhong Yixin Granules groups, with 12 rats in each group. Except for those in the control group, rats in the other groups were induced by intraperitoneal injection of doxorubicin(DOX) into a rat model. After the Luhong Yixin Granules were dissolved in the same amount of normal saline, they were given by gavage at low, medium and high doses(2.8, 5.6, 11.2 g·kg~(-1)·d~(-1)), and the control group and the model group were given the same amount of normal saline by gavage for 40 days. After the end of dosing, echocardiography was used to measure left ventricular ejection fraction(LVEF) and left ventricular fractional shortening(LVFS). Rat body weight(BW) and heart weight(HW) were calculated as HW/BW. Enzyme-linked immunosorbent assay was used to measure the levels of interleukin-6(IL-6), interleukin-17(IL-17), tumor necrosis factor-α(TNF-α), transforming growth factor-β1(TGF-β1), growth stimulation expressed gene 2 protein(ST2), N-terminal pro-B-type natriuretic peptide(NT-proBNP), galectin-3(Gal-3) and creatine kinase isoenzyme(CK-MB) in serum. Hematoxylin-eosin(HE) staining and Masson staining were used to observe the pathological morphology of myocardial tissue. Western blot and quantitative real-time polymerase chain reaction were used to detect the protein and mRNA expression levels of IL-6, IL-17, TNF-α, TGF-β1, Smad3, Smad7, α-smooth muscle actin(α-SMA), and collagen Ⅰ(COL-Ⅰ), respectively. RESULTS:: showed that compared with those in the control group, LVEF, LVFS, and HW/BW in the model group were decreased(P&lt;0.05), and the levels of IL-6, IL-17, TNF-α, TGF-β1, ST2, NT-proBNP, Gal-3, and CK-MB were increased(P&lt;0.05). HE staining showed inflammatory changes in myocardial tissue; Masson staining showed decreases in the cross-sectional area and ventricular cavity area of the heart, and myocardial fibrosis of varying degrees(P&lt;0.05). The protein and mRNA expression of IL-6, IL-17, TNF-α, TGF-β1, Smad3, α-SMA, and COL-Ⅰ were increased(P&lt;0.05), and the protein and mRNA expression of Smad7 protein was decreased(P&lt;0.01). Compared with those in the model group, LVEF, LVFS and HW/BW of the low-, medium-and high-dose Luhong Yixin Granules groups were increased(P&lt;0.05), and the levels of IL-6, IL-17, TNF-α, TGF-β1, ST2, NT-proBNP, Gal-3 and CK-MB were decreased(P&lt;0.05). HE staining showed gradually reduced inflammatory changes of myocardial tissue, and Masson staining showed increased cross-sectional area and ventricular cavity area of the heart and decreased area of myocardial fibrosis(P&lt;0.05). The protein and mRNA expression levels of IL-6, IL-17, TNF-α, TGF-β1, Smad3, α-SMA, and COL-Ⅰ were decreased(P&lt;0.05), while the protein and mRNA expression levels of Smad7 were increased(P&lt;0.05). Luhong Yixin Granules may be of great value in the treatment of heart failure by regulating the TGF-β1/Smads signaling pathway, inhibiting the expression of inflammation-related proteins, reducing the deposition of extracellular matrix, and alleviating myocardial fibrosis.

Animal geneticists and breeders have the impending challenge of enhancing the resilience of Indian livestock to heat stress through better selection strategies. Climate change's impact on livestock is more intense in tropical countries like India where dairy cattle crossbreeds are more sensitive to heat stress. The main reason for this study was to find the missing relative changes in transcript levels in thermo-neutral and heat stress conditions in crossbred cattle through whole-transcriptome analysis of RNA-Seq data. Differentially expressed genes (DEGs) identified based on the minimum log twofold change value and false discovery rate 0.05 revealed 468 up-regulated genes and 2273 down-regulated significant genes. Functional annotation and pathway analysis of these significant DEGs were compared based on Gene Ontology (Biological process), Kyoto Encyclopedia of Genes and Genome (KEGG), and Reactome pathways using g: Profiler, ShinyGO v0.76, and iDEP.951 web tools. On finding network visualization, the most over-represented and correlated pathways were neuronal and sensory organ development, calcium signalling pathway, Mitogen-activated protein kinase (MAPK) and Smad signalling pathway, Ras-proximate-1, or Ras-related protein 1 (Rap 1) signalling pathway, apoptosis, and oxidative stress. Similarly, down-regulated genes were most expressed in mRNA processing, immune system, B-cell receptor signalling pathway, Nucleotide oligomerization domain (NOD)-like receptors (NLRs) signalling pathway and nonsense-mediated decay (NMD) pathway. The heat stress-responsive genes identified in this study will facilitate our understanding of the molecular basis for climate resilience and heat tolerance in Indian dairy crossbreeds., (© 2024. The Author(s) under exclusive licence to International Society of Biometeorology.)

The fruit of Alpinia oxyphylla Miq is an important food spice in southern China and has been used in the treatment of kidney disorders for centuries. In order to discover the natural products with potent renoprotective activities in A. oxyphylla and provide some references for its usage, systematic phytochemical studies were carried out and 24 new diverse sesquiterpenoids, including seven guaiane sesquiterpenoids ( 1 - 7 ), 10 eudesmane sesquiterpenoids ( 9 - 13 , 18 , 19 , and 21 - 23 ), six cadinane sesquiterpenoids ( 31 - 35 and 38 ), and an eremophilane sesquiterpenoid ( 40 ), along with 24 known analogues were isolated and elucidated by analysis of spectroscopic data and quantum-chemical calculations. Biological evaluation showed that 6 sesquiterpenoids could significantly inhibit the expression of extracellular matrix components, α-SMA in TGF-β1 induced kidney proximal tubular cells (NRK-52e) at low concentrations, and 9 sesquiterpenoids could also downregulate fibronectin and collagen I in a concentration-dependent manner, showing their potential in renal fibrosis. Further action mechanism study displayed that TGF-β1/Smads pathway might be involved in the antifibrotic effects of active sesquiterpenoids 15 and 43 . These studies suggest that A. oxyphylla may have a potential to serve as a functional food in preventing renal fibrosis-associated diseases.

Background: Fibrosis is a significant pathological feature of chronic skeletal muscle injury, profoundly affecting muscle regeneration. Fibro-adipogenic progenitors (FAPs) have the ability to differentiate into myofibroblasts, acting as a primary source of extracellular matrix (ECM). the process by which FAPs differentiate into myofibroblasts during chronic skeletal muscle injury remains inadequately explored., Method: mouse model with sciatic nerve denervated was constructed and miRNA expression profiles between the mouse model and uninjured mouse were analyzed. qRT/PCR and immunofluorescence elucidated the effect of miR-27b-3p on fibrosis in vivo and in vitro. Dual-luciferase reporter identified the target gene of miR-27b-3p, and finally knocked down or overexpressed the target gene and phosphorylation inhibition of Smad verified the influence of downstream molecules on the abundance of miR-27b-3p and fibrogenic differentiation of FAPs., Result: FAPs derived from a mouse model with sciatic nerves denervated exhibited a progressively worsening fibrotic phenotype over time. Introducing agomiR-27b-3p effectively suppressed fibrosis both in vitro and in vivo. MiR-27b-3p targeted Transforming Growth Factor Beta Receptor 1 (TGF-βR1) and the abundance of miR-27b-3p was negatively regulated by TGF-βR1/Smad., Conclusion: miR-27b-3p targeting the TGF-βR1/Smad pathway is a novel mechanism for regulating fibrogenic differentiation of FAPs. Increasing abundance of miR-27b-3p, suppressing expression of TGF-βR1 and inhibiting phosphorylation of smad3 presented potential strategies for treating fibrosis in chronic skeletal muscle injury., (© 2024. The Author(s).)

This study investigated the effects of modified Fangji Huangqi Decoction on the expression of proteins related to epithelial-mesenchymal transition(EMT) in a mouse model of unilateral ureteral obstruction( UUO) and in a rat renal tubular epithelial cell(NRK-52E) model of fibrosis induced by transforming growth factor β1(TGF-β1). It aims to decipher the molecular mechanism by which modified Fangji Huangqi Decoction alleviates renal interstitial fibrosis. C57/BL mice were subjected to UUO.After the surgery, the mice were treated with 0. 5-fold and 2-fold concentrations of modified Fangji Huangqi Decoction and fosinopril sodium(positive control) for 7 days. The interstitial collagen deposition in the kidney was assessed by Masson staining. Western blot and RT-qPCR were employed to determine the expression levels of TGF-β1, phosphorylated Smad2/3(p-Smad2/3), Smad2/3, Snail,epithelial cadherin(E-cadherin), alpha smooth muscle actin(α-SMA), and vimentin. The NRK-52E cell model induced by TGF-β1was treated with the serum samples collected from SD rats treated with different concentrations of modified Fangji Huangqi Decoction.The CCK-8 assay was employed to examine the effects of the serum samples on NRK-52E cell proliferation. The cell morphology in different groups was observed under a microscope. Furthermore, the modeled cells were treated with the serum containing 1-fold decoction. Western blot and RT-qPCR were then employed to measure the expression levels of p-Smad2/3, Smad2/3, Snail,E-cadherin, α-SMA, and vimentin in the cells. Under the same conditions, sh RNA was used to silence the Snail gene, and measurements were repeated before and after treatment with the serum containing 1-fold decoction. The results indicated that modified Fangji Huangqi Decoction alleviated the fibrotic injury in the mouse model of UUO and the fibrosis in the NRK-52E cell model. The treatment with the decoction down-regulated the protein and m RNA levels of EMT-related indicators including p-Smad2/3, α-SMA,Snail, and vimentin, while it up-regulated the expression of E-cadherin. After sh RNA silencing of the Snail gene, the protein and m RNA levels of E-cadherin, α-SMA, and vimentin showed no significant differences before and after treatment with the serum containing the decoction. The results suggest that modified Fangji Huangqi Decoction may alleviate renal interstitial fibrosis by inhibiting the TGF-β1/Smad/Snail signaling pathway and regulating the EMT process.

Acute myeloid leukemia (AML) is one of the most prevalent types of leukemia and is challenging to cure for most patients. Basic Leucine Zipper ATF-Like Transcription Factor (BATF) has been reported to participate in the development and progression of numerous tumors. However, its role in AML is largely unknown. In this study, the expression and prognostic value of BATF were examined in AML. Our results demonstrated that BATF expression was upregulated in AML patients, which was significantly correlated with poor clinical characteristics and survival. Afterward, functional experiments were performed after knocking down or overexpressing BATF by transfecting small interfering RNAs and overexpression plasmids into AML cells. Our findings revealed that BATF promoted the migratory and invasive abilities of AML cells in vitro and in vivo. Moreover, the target genes of BATF were searched from databases to explore the binding of BATF to the target gene using ChIP and luciferase assays. Notably, our observations validated that BATF is bound to the promoter region of TGF-β1, which could transcriptionally enhance the expression of TGF-β1 and activate the TGF-β1/Smad/MMPs signaling pathway. In summary, our study established the aberrantly high expression of BATF and its pro-migratory function via the TGF-β1-Smad2/3-MMP2/9 axis in AML, which provides novel insights into extramedullary infiltration of AML., (© 2024 The Authors. Molecular Carcinogenesis published by Wiley Periodicals LLC.)

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-βR2) 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-βR2 interactions may exert scars and nerves functions and suggest potential therapeutic targets for treating spinal fibrotic diseases., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing financial interests or personal relationships that may have influenced the work reported in this study., (Copyright © 2024. Published by Elsevier Ltd.)

Breast cancer is a serious threat to human health. The transforming growth factor-β signaling pathway is an important pathway involved in the occurrence and development of cancer. The SMAD family genes are responsible for the TGF-β signaling pathway. However, the mechanism by which genes of the SMAD family are involved in breast cancer is still unclear. Therefore, it is necessary to investigate the biological roles of the SMAD family genes in breast cancer. We downloaded the gene expression data, gene mutation data, and clinical pathological data of breast cancer patients from the UCSC Xena database. We used the Wilcox test to estimate the expression of genes of the SMAD family in cancers. And the biological functions of SMAD family genes using the DAVID website. The Pearson correlation method was used to explore the immune cell infiltration and drug response of SMAD family genes. We conducted in biological experiments vitro and vivo. In this study, we integrated the multi-omics data from TCGA breast cancer patients for analysis. The expression of genes of SMAD family was significantly dysregulated in patients with breast cancer. Except for SMAD6, the expression of other SMAD family genes was positively correlated. We also found that genes of the SMAD family were significantly enriched in the TGF-β signaling pathway, Hippo signaling pathway, cell cycle, and cancer-related pathways. In addition, SMAD3, SMAD6, and SMAD7 were lowly expressed in stage II breast cancer, while SMAD4 and SMAD2 were lowly expressed in stage III cancer. Furthermore, the expression of genes of the SMAD family was significantly correlated with immune cell infiltration scores. Constructing a xenograft tumor mouse model, we found that SMAD3 knockdown significantly inhibited tumorigenesis. Finally, we analyzed the association between these genes and the IC50 value of drugs. Interestingly, patients with high expression of SMAD3 exhibited significant resistance to dasatinib and staurosporine, while high sensitivity to tamoxifen and auranofin. In addition, SMAD3 knockdown promoted the apoptosis of BT-549 cells and decreased cell activity, and BAY-1161909 and XK-469 increased drug efficacy. In conclusion, genes of the SMAD family play a crucial role in the development of breast cancer., (© 2024. The Author(s).)

Liver fibrosis refers to the excessive buildup of extracellular matrix (ECM) components in liver tissue. It is considered a pathological response to liver damage for which there is no effective treatment. Aloin, an anthraquinone compound isolated from the aloe plant, has shown good pharmacological effects in the treatment of gastric cancer, ulcerative colitis, myocardial hypertrophy, traumatic brain injury, and other diseases; however, its specific impact on liver fibrosis remains unclear. To address this gap, we conducted a study to explore the mechanisms underlying the potential antifibrotic effect of aloin. We constructed a mouse liver fibrosis model using carbon tetrachloride (CCl 4 ) dissolved in olive oil as a modeling drug. Additionally, a cellular model was developed by using transforming growth factor β1 (TGF-β1) as a stimulus applied to hepatic stellate cells. After aloin intervention, serum alanine aminotransferase, hepatic hydroxyproline, and serum aspartate aminotransferase were reduced in mice after aloin intervention compared to CCl 4 -mediated liver injury without aloin intervention. Aloin relieved the oxidative stress caused by CCl 4 via reducing hepatic malondialdehyde in liver tissue and increasing the level of superoxide dismutase. Aloin treatment decreased interleukin (IL)-1β, IL-6, and tumor necrosis factor-α and increased the expression of IL-10, which inhibited the inflammatory response in liver injury. In addition, aloin inhibited the activation of hepatic stellate cells and reduced the level of α-smooth muscle actin (α-SMA) and collagen type I. In cell and animal experiments, aloin attenuated liver fibrosis, acting through the TGF-β/Smad2/3 signaling pathway, and mitigated CCl 4 - and TGF-β1-induced inflammation. Thus, the findings of this study provided theoretical data support and a new possible treatment strategy for liver fibrosis.

Background and Aims: Liver fibrosis is a leading indicator for increased mortality and long-term comorbidity in NASH. Activation of HSCs and excessive extracellular matrix production are the hallmarks of liver fibrogenesis. Tyrosine kinase receptor (TrkB) is a multifunctional receptor that participates in neurodegenerative disorders. However, paucity of literature is available about TrkB function in liver fibrosis. Herein, the regulatory network and therapeutic potential of TrkB were explored in the progression of hepatic fibrosis., Methods and Results: The protein level of TrkB was decreased in mouse models of CDAHFD feeding or carbon tetrachloride-induced hepatic fibrosis. TrkB suppressed TGF-β-stimulated proliferation and activation of HSCs in 3-dimensional liver spheroids and significantly repressed TGF-β/SMAD signaling pathway either in HSCs or in hepatocytes. The cytokine, TGF-β, boosted Nedd4 family interacting protein-1 (Ndfip1) expression, promoting the ubiquitination and degradation of TrkB through E3 ligase Nedd4-2. Moreover, carbon tetrachloride intoxication-induced hepatic fibrosis in mouse models was reduced by adeno-associated virus vector serotype 6 (AAV6)-mediated TrkB overexpression in HSCs. In addition, in murine models of CDAHFD feeding and Gubra-Amylin NASH (GAN), fibrogenesis was reduced by adeno-associated virus vector serotype 8 (AAV8)-mediated TrkB overexpression in hepatocytes., Conclusion: TGF-β stimulated TrkB degradation through E3 ligase Nedd4-2 in HSCs. TrkB overexpression inhibited the activation of TGF-β/SMAD signaling and alleviated the hepatic fibrosis both in vitro and in vivo . These findings demonstrate that TrkB could be a significant suppressor of hepatic fibrosis and confer a potential therapeutic target in hepatic fibrosis., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Diabetic kidney disease is an important microvascular complication of diabetes and the leading cause of end-stage renal disease. Its pathological characteristics mainly include epithelial mesenchymal transition(EMT) in glomerulus, podocyte apoptosis and autophagy, and damage of glomerular filtration barrier. Transforming growth factor-β(TGF-β)/Smad signaling pathway is specifically regulated by a variety of mechanisms, and is a classic pathway involved in physiological activities such as apoptosis, proliferation and differentiation. At present, many studies have found that TGF-β/Smad signaling pathway plays a key role in the pathogenesis of diabetic kidney disease. Traditional Chinese medicine has significant advantages in the treatment of diabetic kidney disease for its multi-component, multi-target and multi-pathway characteristics, and some traditional Chinese medicine extracts, traditional Chinese medicines and traditional Chinese medicine compound prescription improve the renal injury of diabetic kidney disease by regulating TGF-β/Smad signaling pathway. This study clarified the mechanism of TGF-β/Smad signaling pathway in diabetic kidney disease by expounding the relationship between the key targets of the pathway and diabetic kidney disease, and summarized the research progress of traditional Chinese medicine in the treatment of diabetic kidney disease by interfering with TGF-β/Smad signaling pathway in recent years, to provide reference for drug research and clinical treatment of diabetic kidney disease in the future.

Ankylosing spondylitis (AS) is a refractory autoimmune disease, whose typical pathology is the development of inflammation to ossification and ankylosis. Histone deacetylase 1 (HDAC1) is considered to be a key factor involved in inflammatory gene transduction, but its role in AS remains unclear. The purpose of this study was to explore the role and possible mechanism of HDAC1 in AS based on the Wnt-Smad pathway. Fibroblasts were isolated from hip synovial tissues of AS patients, adeno-associated virus (AAV) was used to regulate the expression of HDAC1, DKK-1 and SIS3 was used to inhibit Wnt and Smad, respectively. The expressions of Wnt-Smad pathway-related proteins were analyzed by WB, and the TRP ion channel proteins were analyzed by immunofluorescence and WB. The proliferation of AS fibroblasts was detected by CCK-8, the expression of inflammatory cytokines was detected by ELISA, and the effects of HDAC1 on osteogenic differentiation of AS fibroblasts were investigated by alkaline phosphatase (ALP) activity, intracellular calcium concentration, mineralization and osteogenic proteins expressions. Results showed that HDAC1 significantly affected the protein expressions of the Wnt-Smad pathway in AS fibroblasts, and Wnt inhibitor DKK-1 and Smad3 inhibitor SIS3 could significantly reverse the effect of HDAC1 on the Wnt-Smad pathway. In addition, HDAC1 significantly activated the TRP ion channel and promoted the proliferation, inflammatory response and osteogenic differentiation of AS fibroblasts. DKK-1 or SIS3 treatment significantly inhibit the effect of HDAC-1 on AS fibroblasts, suggesting that the Wnt-Smad pathway is involved in the regulation of AS by HDAC1. In conclusion, HDAC1 promotes the proliferation, inflammatory response and osteogenic differentiation of AS fibroblasts through the Wnt-Smad pathway., (© 2022. The Author(s).)

Cyclophilin D (CypD) promotes opening of the mitochondrial permeability transition pore (MPTP) which plays a key role in both cell physiology and pathology. It is, therefore, beneficial for cells to tightly regulate CypD and MPTP but little is known about such regulation. We have reported before that CypD is downregulated and MPTP deactivated during differentiation in various tissues. Herein, we identify BMP/Smad signaling, a major driver of differentiation, as a transcriptional regulator of the CypD gene, Ppif . Using osteogenic induction of mesenchymal lineage cells as a BMP/Smad activation-dependent differentiation model, we show that CypD is in fact transcriptionally repressed during this process. The importance of such CypD downregulation is evidenced by the negative effect of CypD 'rescue' via gain-of-function on osteogenesis both in vitro and in a mouse model. In sum, we characterized BMP/Smad signaling as a regulator of CypD expression and elucidated the role of CypD downregulation during cell differentiation., Competing Interests: RS, BK, KE, JJ, GP, HA, RE No competing interests declared, (© 2022, Sautchuk et al.)

The histone methyltransferase SET domain bifurcated 1 (SETDB1) catalyzes the trimethylation of lysine 9 of histone H3, thereby regulating gene expression. In this study, we used conditional knockout mice, where Setdb1 was deleted only in neural crest cells (Setdb1 fl/fl, Wnt1-Cre +  mice), to clarify the role of SETDB1 in palatal development. Setdb1 fl/fl, Wnt1-Cre +  mice died shortly after birth due to a cleft palate with full penetration. Reduced palatal mesenchyme proliferation was seen in Setdb1 fl/fl, Wnt1-Cre +  mice, which might be a possible mechanism of cleft palate development. Quantitative RT-PCR and in situ hybridization showed that expression of the Pax9, Bmp4, Bmpr1a, Wnt5a, and Fgf10 genes, known to be important for palatal development, were markedly decreased in the palatal mesenchyme of Setdb1 fl/fl, Wnt1-Cre +  mice. Along with these phenomena, SMAD1/5/9 phosphorylation was decreased by the loss of Setdb1. Our results demonstrated that SETDB1 is indispensable for palatal development partially through its proliferative effect. Taken together with previous reports that PAX9 regulates BMP signaling during palatal development which implies that loss of Setdb1 may be involved in the cleft palate development by decreasing SMAD-dependent BMP signaling through Pax9., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Ethnopharmacological Relevance: Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease with unknown etiology. Oxytropis falcata Bunge (O. falcata) is a 1-35 cm high perennial clustered herb, also known as edaxia, has viscosity and a special smell, and is mainly distributed in the western areas of China. The root of O. falcata has a diameter of 6 mm, is straight and deep, dark red and its stems are shortened, woody and multibranched. O. falcata has heat-clearing, detoxification, analgesic, anti-inflammatory, antibacterial, hemostatic and antitumor activities. Furthermore, O. falcata has excellent anti-inflammatory and analgesic effects, and it is one of the three major anti-inflammatory drugs in Tibetan medicine, known as "the king of herbs". Total flavonoids of Oxytropis falcata Bunge (FOFB) were previously extracted, and their pharmacological activities are consistent with those of the whole herb. In this study, FOFB was extracted from O. falcata by ethanol extraction, and the mechanism of FOFB on IPF was verified by in vivo and in vitro experiments., Aim of the Study: In this study, we aimed to observe the effects of FOFB on idiopathic pulmonary fibrosis., Materials and Methods: In in vivo experiments, an IPF rat model was established by bleomycin induction. The rats were treated with FOFB (100, 200, 400 mg kg -1 ·d -1 ) for 4 weeks. Masson staining and the expression of TGF-β, p-Smad2, p-Smad3 and Smad7 in the lung tissue of rats were detected. In in vitro experiments, we perfused normal rats with FOFB (100, 200, 400 mg kg -1 ·d -1 ) and obtained the corresponding drug-containing serum. The HFL-1 cell model induced by TGF-β1 was used to detect the corresponding indices through intervention with drug-containing serum. The best intervention time for drug-containing serum was detected by the CCK-8 method. Changes in apoptosis, cytoskeleton and rough endoplasmic reticulum structure were detected. Finally, the expression of TGF-β, p-Smad2, p-Smad3 and Smad7 in cells was examined., Results: In vivo, Masson staining indicated that the degree of pulmonary fibrosis increased significantly, the expression of TGF-β, p-smad2 and p-Smad3 increased significantly, and the expression of Smad7 decreased in the model group. We found that the degree of pulmonary fibrosis gradually decreased and that the inhibition of the TGF-β/Smad signaling pathway became more obvious with increasing FOFB dose. FOFB (400 mg kg -1 ·d -1 ) significantly improved the degree of pulmonary fibrosis in rats. In in vitro experiments, the CCK-8 results showed that 120 h was the best intervention time for drug-containing serum. In the model group, there was no obvious apoptosis or changes in microfilaments and microtubules, the number of rough endoplasmic reticulum increased, and the expression of TGF-β, p-Smad2 and p-Smad3 increased significantly, while the expression of Smad7 decreased significantly. We found that with the increase in drug-containing serum concentration, the apoptosis, cytoskeleton and degree of destruction of the rough endoplasmic reticulum in the HFL-1 cell model also increased, and the inhibition of the TGF-β/Smad signaling pathway became more pronounced; the effect of the drug-containing serum administered with FOFB (400 mg kg -1 ·d -1 ) was the most significant., Conclusions: The results suggest that FOFB can improve the occurrence and development of IPF. The effect of FOFB on IPF may be mediated by inhibition of the TGF-β1/Smad signaling pathway., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Nerve growth factor-induced gene B (Nur77) has been shown to ameliorate several biological processes in chronic diseases, including inflammatory response, cellular proliferation, and metabolism. Chronic kidney disease (CKD) is characterized by tubulointerstitial fibrosis for which no targeted therapies are available as yet. In this study, we performed in vivo and in vitro experiments to demonstrate that Nur77 targets fibrosis signals and attenuates renal tubulointerstitial fibrosis during the aging process. We observed that the TGF-β/Smads signal pathway was significantly suppressed by Nur77, suggesting that Nur77 controlled the activation of key steps in TGF-β/Smads signaling. We further showed that Nur77 interacted with Smad7, the main repressor of nuclear translocation of Smad2/3, and stabilized Smad7 protein homeostasis. Nur77 deficiency resulted in Smad7 degradation, aggravating Smad2/3 phosphorylation, and promoting transcription of its downstream target genes, ACTA2 and collagen I. Our findings demonstrate that Nur77 is a potential therapeutic target for age-related kidney diseases including CKD. Maintenance of Nur77 may be an effective strategy for blocking renal tubulointerstitial fibrosis and improving renal function in the elderly., (© 2021 Federation of American Societies for Experimental Biology.)

TGFβ signaling promotes progression of bone-metastatic (BMET) breast cancer (BCa) cells by driving tumor-associated osteolysis, a hallmark of BCa BMETs, thus allowing for tumor expansion within bone. Turmeric-derived bioactive curcumin, enriched in bone via local enzymatic deconjugation of inactive circulating curcumin-glucuronides, inhibits osteolysis and BMET progression in human xenograft BCa BMET models by blocking tumoral TGFβ signaling pathways mediating osteolysis. This is a unique antiosteolytic mechanism in contrast to current osteoclast-targeting therapeutics. Therefore, experiments were undertaken to elucidate the mechanism for curcumin inhibition of BCa TGFβ signaling and the application of this finding across multiple BCa cell lines forming TGFβ-dependent BMETs, including a possible role for bioactive curcumin metabolites in mediating these effects. Immunoblot analysis of TGFβ signaling proteins in bone tropic human (MDA-SA, MDA-1833, MDA-2287) and murine (4T1) BCa cells revealed uniform curcumin blockade of TGFβ-induced Smad activation due to down-regulation of plasma membrane associated TGFβR2 and cellular receptor Smad proteins that propagate Smad-mediated gene expression, resulting in downregulation of PTHrP expression, the osteolytic factor driving in vivo BMET progression. With the exception of early decreases in TGFβR2, inhibitory effects appeared to be mediated by oxidative metabolites of curcumin and involved inhibition of gene expression. Interestingly, while not contributing to changes in Smad-mediated TGFβ signaling, curcumin caused early activation of MAPK signaling in all cell lines, including JNK, an effect possibly involving interactions with TGFβR2 within lipid rafts. Treatment with curcumin or oxidizable analogs of curcumin may have clinical relevancy in the management of TGFβ-dependent BCa BMETs., (Copyright © 2021. Published by Elsevier Inc.)

Patulin (PAT) is a mycotoxin that contaminates a variety of food and foodstuffs. Earlier in vitro and in vivo findings have indicated that kidney is one of the target organs for PAT-induced toxicity. However, no study has evaluated the chronic effects of PAT exposure at environmentally relevant doses or elucidated the detailed mechanism(s) involved. Here, using in vitro and in vivo experimental approaches, we delineated the mechanism/s involved in pro-fibrotic changes in the kidney after low-dose chronic exposure to PAT. We found that non-toxic concentrations (50 nM and 100 nM) of PAT to normal rat kidney cells (NRK52E) caused a higher generation of reactive oxygen species (ROS) (mainly hydroxyl (•OH), peroxynitrite (ONOO - ), and hypochlorite radical (ClO-). PAT exposure caused the activation of mitogen-activated protein kinases (MAPKs) and its downstream c-Jun/Fos signaling pathways. Moreover, our chromatin immunoprecipitation (ChIP) analysis suggested that c-Jun/Fos binds to the promoter region of Transforming growth factor beta (TGF-β 1 ) and possibly induces its expression. Results showed that PAT-induced TGF-β 1 further activates the TGF-β 1 /smad signaling pathways. Higher activation of slug and snail transcription factors further modulates the regulation of pro-fibrotic molecules. Similarly, in vivo results showed that PAT exposure to rats through gavage at 25 and 100 μg/kg b. wt had higher levels of kidney injury/toxicity markers namely vascular endothelial growth factor (VEGF), kidney Injury Molecule-1 (Kim-1), tissue inhibitor of metalloproteinase-1 (Timp-1), and clusterin (CLU). Additionally, histopathological analysis indicated significant alterations in renal tubules and glomeruli along with collagen deposition in PAT-treated rat kidneys. Overall, our data provide evidence of the involvement of ROS mediated MAPKs and TGF-β 1 /smad pathways in PAT-induced pro-fibrotic changes in the kidney via modulation of slug and snail expression., (Copyright © 2021. Published by Elsevier Inc.)

This study aims to investigate the effects of exposure to different dosages of zearalenone (ZEA) on cecal physical barrier functions and its mechanisms based on the TGF-β1/Smads signaling pathway in weaned piglets. Thirty-two weaned piglets were allotted to four groups and fed a basal diet supplemented with ZEA at 0, 0.15, 1.5, and 3.0 mg/kg, respectively. The results showed that 1.5 and 3.0 mg/kg ZEA damaged cecum morphology and microvilli, and changed distribution and shape of M cells. Moreover, 1.5 and 3.0 mg/kg ZEA decreased numbers of goblet cells, the expressions of TFF3 and tight junction proteins, and inhibited the TGF-β1/Smads signaling pathway. Interestingly, the 0.15 mg/kg ZEA had no significant effect on cecal physical barrier functions but decreased the expressions of Smad3, p-Smad3 and Smad7. Our study suggests that high-dose ZEA exposure impairs cecal physical barrier functions through inhibiting the TGF-β1/Smads signaling pathway, but low-dose ZEA had no significant effect on cecum morphology and integrity through inhibiting the expression of smad7. These findings provide a scientific basis for helping people explore how to reduce the toxicity of ZEA in feeds.

This study explores the effect of acute Ethanol (EtOH) exposure on Bone Morphogenetic Protein (BMP)-evoked intracellular signaling, and the concomitant morphological changes induced by EtOH in C2C12 cells and DRG (Dorsal root ganglion) neurons in an in vitro model related to Fetal Alcohol Syndrome Disorder (FASD). All assays were performed within 30 min of BMP stimulation to specifically investigate the earliest events occurring in BMP-evoked intracellular signaling pathways. We show that Smad phosphorylation and nuclear translocation stimulated by BMPs was not altered following acute exposure to EtOH. In contrast, acute EtOH exposure alone caused a striking concentration-dependent decrease in Akt phosphorylation, as well as a loss of adhesion in C2C12 cells. The addition of BMPs before exposure to EtOH was associated with maintenance of Akt phosphorylation, greater cell adhesion in C2C12 cells, and preservation of growth cone complexity in DRG neurons. Thus, for both C2C12 cells and DRG neurons, BMPs, acting through non-canonical BMP signaling pathways, appear to impart some protection against the profound effects of acute EtOH exposure on cellular adhesion and structure., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that would influence this study., (Copyright © 2021 Elsevier B.V. All rights reserved.)

This study investigated the role of microRNA (miRNA) miR-486-5p in oxidative stress injury in hepatocytes under the treatment of mesenchymal stem cell conditioned medium (MSC-CM). The oxidative stress injury in hepatocytes (L02) was induced by H 2 O 2 . Human umbilical cord blood MSC-CM (UCB-MSC-CM) was prepared. The effects of UCB-MSC-CM on the proliferation, apoptosis, and inflammatory response in L02 cells were detected by Cell Counting Kit-8 (CCK-8) assay, flow cytometry analysis, and enzyme-linked immunosorbent assay (ELISA). Subsequently, the target of miR-486-5p was predicted using bioinformatics analysis, and the possible signaling pathway addressed by miR-486-5p was explored using western blot. We found that miR-486-5p expression was elevated following oxidative stress injury and was reduced after UCB-MSC-CM treatment. UCB-MSC-CM protected L02 cells against H 2 O 2 -induced injury by downregulation of miR-486-5p. Proviral integration site for Moloney murine leukemia virus 1 (PIM1) was verified to be targeted by miR-486-5p. UCB-MSC-CM upregulated the expression of PIM1 reduced by H 2 O 2 in L02 cells. Additionally, silencing PIM1 attenuated the protective effects of miR-486-5p downregulation against oxidative stress injury. We further demonstrated that UCB-MSC-CM inhibited the TGF-β/Smad signaling in H 2 O 2- treated L02 cells by the miR-486-5p/PIM1 axis. Overall, UCB-MSC-CM attenuates oxidative stress injury in hepatocytes by downregulating miR-486-5p and upregulating PIM1, which may be related to the inhibition of TGF-β/Smad pathway.

The Angiotensin II/transforming growth factor-β1 (AngII/TGF-β1) signal axis is an important regulatory pathway for atrial fibrosis, which can contribute to atrial fibrillation (AF). Fused in sarcoma (FUS) was recently found to regulate cardiac diseases. This study aimed to investigate whether FUS could regulate AngII induced fibrosis and uncover the possible mechanisms. The expression of FUS in AF patients and AngII-induced cardiac fibroblasts was measured by RT-qPCR and western blot assays. Fus was silenced in cells using short hairpin RNA (shRNA), then cell proliferation, migration, collagen synthesis and TGF-β1/Smad signaling were detected by CCK-8, wound healing and western blot assays, respectively. The possible target for Fus was predicted by searching Starbase database and verified by RNA-binding protein immunoprecipitation (RIP) and RNA pull down. Cells were overexpressed with Pax3 in the presence of Fus silence and AngII stimulation, then the above cellular processes were further evaluated. Results showed that FUS was upregulated in AF patients and AngII-induced cardiac fibroblasts. Fus knockdown inhibited AngII-enhanced cell proliferation, migration, collagen synthesis and TGF-β1/Smad signaling activation. Furthermore, Fus functions as an RNA-binding protein to bind to Pax3 mRNA and positively regulate its expression. Further studies demonstrated that Pax3 overexpression canceled the above effects of Fus knockdown on cell proliferation, migration, collagen synthesis, and TGF-β1/Smad signaling activation in AngII-induced cells. In conclusion, Fus could target Pax3 to increase the pro-fibrotic effect of AngII in cardiac fibroblasts via activating TGF-β1/Smad signaling. Knockdown of Fus/Pax3 axis may provide a potential therapy for relieving AF.

Background: Hepatocellular carcinoma (HCC) is one of the most challenging malignancies, with high morbidity and mortality rates. The transforming growth factor-β (TGF-β) pathway plays a dual role in HCC, acting as both tumor suppressor and promoter. A thorough understanding of the mechanisms underlying its opposing functions is important. The growth suppressive effects of TGF-β remain largely unknown for mesenchymal HCC cells. Using a systematic approach, here we assess the cytostatic TGF-β responses and intracellular transduction of the canonical TGF-β/Smad signaling cascade in mesenchymal-like HCC cell lines., Methods: Nine mesenchymal-like HCC cell lines, including SNU182, SNU387, SNU398, SNU423, SNU449, SNU475, Mahlavu, Focus, and Sk-Hep1, were used in this study. The cytostatic effects of TGF-β were evaluated by cell cycle analysis, BrdU labeling, and SA-β-Gal assay. RT-PCR and western blot analysis were utilized to determine the mRNA and protein expression levels of TGF-β signaling components and cytostatic genes. Immunoperoxidase staining and luciferase reporter assays were performed to comprehend the transduction of the canonical TGF-β pathway., Results: We report that mesenchymal-like HCC cell lines are resistant to TGF-β-induced growth suppression. The vast majority of cell lines have an active canonical signaling from the cell membrane to the nucleus. Three cell lines had lost the expression of cytostatic effector genes., Conclusion: Our findings reveal that cytostatic TGF-β responses have been selectively lost in mesenchymal-like HCC cell lines. Notably, their lack of responsiveness was not associated with a widespread impairment of TGF-β signaling cascade. These cell lines may serve as valuable models for studying the molecular mechanisms underlying the loss of TGF-β-mediated cytostasis during hepatocarcinogenesis., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Gastric cancer is the fifth most common malignancy in the world with alow 5-year survival rate. To date, no study has investigated the prognostic role of the small mother against decapentaplegic (SMAD) in gastric cancer. The association of SMADs with overall survival (OS) of gastric cancer was analyzed on the online Kaplan-Meier (KM) plotter database. Clinical data such as stage, differentiation, gender, treatment, and Her2 mutation status of gastric cancer patients were analyzed. The (E)-SIS3 was used to inhibit SMAD3 expression in gastric cancer cells, and the effects of SMAD3 on gastric cancer cells were analyzed via real-time cellular analysis (RTCA), flow cytometry, colony formation, and immunofluorescence assay. The results showed that the high expression of three members of SMADs (SMAD1, SMAD2, SMAD4) was correlated with afavorable OS of gastric cancer patients. Meanwhile, SMAD3 expression level indicated highly differentiated cancer. We also observed that surgical treatment was associated with high expression level of SMAD1 and SMAD2. Besides, the effect of Her2 on gastric cancer was not noticeable. Moreover, (E)-SIS3 pharmacological assay revealed that inhibition of expression of SMAD3 suppressed the proliferation and migration ability of gastric cancer cells via inducing apoptosis. Collectively, these results demonstrate that the high expression level of three members of SMADs (SMAD1, SMAD2, and SMAD4) is significantly correlated with favorable OS of gastric cancer patients, which is opposite to SMAD3. Thus, SMADs regulate the differentiation of cancer and can be used to guide treatment decisions.

Subretinal fibrosis is a key pathological feature in neovascular age-related macular degeneration (nAMD). Previously, we identified soluble very low-density lipoprotein receptor (sVLDLR) as an endogenous Wnt signaling inhibitor. This study investigates whether sVLDLR plays an anti-fibrogenic role in nAMD models, including Vldlr -/- mice and laser-induced choroidal neovascularization (CNV). We found that fibrosis factors including P-Smad2/3, α-SMA, and CTGF were upregulated in the subretinal area of Vldlr -/- mice and the laser-induced CNV model. The antibody blocking Wnt co-receptor LRP6 significantly attenuated the overexpression of fibrotic factors in these two models. Moreover, there was a significant reduction of sVLDLR in the interphotoreceptor matrix (IPM) in the laser-induced CNV model. A transgenic strain (sVLDLR-Tg) with sVLDLR overexpression in the IPM was generated. Overexpression of sVLDLR ameliorated the profibrotic changes in the subretinal area of the laser-induced CNV model. In addition, Wnt and TGF-β signaling synergistically promoted fibrogenesis in human primary retinal pigment epithelium (RPE) cells. CRISPR/Cas9-mediated LRP6 gene knockout (KO) attenuated this synergistic effect. The disruption of VLDLR expression promoted, while the overexpression of sVLDLR inhibited TGF-β-induced fibrosis. These findings suggest that overactivated Wnt signaling enhances the TGF-β pathway in subretinal fibrosis. sVLDLR confers an antifibrotic effect, at least partially, through the inhibition of Wnt signaling and thus, has therapeutic potential for fibrosis., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

The successful treatment of keloids is a great challenge in the plastic surgery field. Activating transcription factor 3 (ATF3) is discovered as an adaptive responsive gene, which plays a critical role in fibroblast activation. This study aimed to investigate the expression and biological role of ATF3 in the pathogenesis of keloids. ATF3 expression in normal skins and keloids was evaluated by real-time PCR, western blot and immunohistochemistry. Effects of ATF3 on cell growth, apoptosis, invasion and collagen production were evaluated in keloid fibroblast cells overexpressing or downregulating ATF3. ATF3 expression was significantly elevated in keloid tissues when compared with that of normal skins and parakeloidal skin tissues. Moreover, ATF3 promoted cell proliferation and collagen production in keloid fibroblast cells. Conversely, transfection with siRNA targeting ATF3 led to decreased cell viability and collagen synthesis via inhibiting transforming growth factor-β1 (TGF-β1) and fibroblast growth factor 2/8 (FGF2/8) production in keloid fibroblasts. ATF3 could reduce the apoptosis rate of keloid fibroblast cells. Molecularly, we found that ATF3 promoted BCL2 level and inhibit the expression of BCL2 associated agonist of cell death (Bad), Caspase3 and Caspase9 in keloid fibroblast cells. ATF3 also enhanced the invasive potential via upregulating the expression of Matrix Metalloproteinases (MMP) family members (MMP1, MMP2, MMP9 and MMP13). ATF3 could induce activation of TGF-β/Smad signaling pathway in fibroblasts. Collectively, ATF3 could promote growth and invasion, and inhibit apoptosis via TGF-β/Smad pathway in keloid fibroblast cells, suggesting that ATF3 might be considered as a novel therapeutic target for the management of keloid.

Liver fibrosis is caused by repetitive hepatic injury. Regulated in development and DNA damage response 1 (REDD1) gene is induced by various stresses and has been studied in cell proliferation and survival. However, the role of REDD1 in hepatic stellate cell activation and hepatic fibrogenesis has not yet been investigated. In the current study, we examined the effect of REDD1 on hepatic fibrogenesis and the underlying molecular mechanism. REDD1 protein was upregulated in the activated primary hepatic stellate cells and transforming growth factor-β (TGF-β)-treated LX-2 cells. REDD1 mRNA levels were also elevated by TGF-β treatment. TGF-β signaling is primarily transduced via the activation of the Smad transcription factor. However, TGF-β-mediated REDD1 induction was not Smad-dependent. Thus, we investigated the transcription factors that influence the REDD1 expression by TGF-β. We found that c-JUN, a component of AP-1, upregulated the REDD1 expression that was specifically suppressed by p38 inhibitor. In silico analysis of the REDD1 promoter region showed putative AP-1-binding sites; additionally, its deletion mutants demonstrated that the AP-1-binding site between -716 and -587 bp within the REDD1 promoter is critical for TGF-β-mediated REDD1 induction. Moreover, REDD1 overexpression markedly inhibited TGF-β-induced plasminogen activator inhibitor-1 (PAI-1) expression and Smad phosphorylation. REDD1 adenovirus infection inhibited CCl 4 -induced hepatic injury in mice, which was demonstrated by reduced ALT/AST levels and collagen accumulation. In addition, we observed that REDD1 inhibited CCl 4 -induced fibrogenic gene induction and restored GSH and malondialdehyde levels. Our findings implied that REDD1 has the potential to inhibit HSC activation and protect against liver fibrosis., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Over the last few years, the Transforming growth factor- β (TGF-β) has been significantly considered as an effective and ubiquitous mediator of cell growth. The cytokine, TGF-β is being increasingly recognized as the most potent inducer of cancer cell initiation, differentiation, migration as well as progression through both the SMAD-dependent and independent pathways. There is growing evidence that supports the role of secretory cytokine TGF-β as a crucial mediator of tumor-stroma crosstalk. Contextually, the CAFs are the prominent component of tumor stroma that helps in tumor progression and onset of chemoresistance. The interplay between the CAFs and the tumor cells through the paracrine signals is facilitated by cytokine TGF-β to induce the malignant progression. Here in this review, we have dissected the most recent advancements in understanding the mechanisms of TGF-β induced CAF activation, their multiple origins, and most importantly their role in conferring chemoresistance. Considering the pivotal role of TGF-β in tumor perogression and associated stemness, it is one the proven clinical targets We have also included the clinical trials going on, targeting the TGF-β and CAFs crosstalk with the tumor cells. Ultimately, we have underscored some of the outstanding issues that must be deciphered with utmost importance to unravel the successful strategies of anti-cancer therapies., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Radiation‑induced lung tissue injury is an important reason for the limited application of radiotherapy on thoracic malignancies. Previously, we reported that administration of Jiawei‑Maxing‑Shigan decoction (JMSD) attenuated the radiation‑induced epithelial‑mesenchymal transition (EMT) in alveolar epithelial cells (AECs) via TGF‑β/Smad signaling. The present study aimed to examine the role of protein phosphatase Mg 2+ /Mn 2+ ‑dependent 1A (PPM1A) in the anti‑EMT activity of JMSD on AECs. The components in the aqueous extract of JMSD were identified by high‑performance liquid chromatography coupled with electrospray mass spectrometry. Primary rat type II AECs were treated with radiation (60Co γ‑ray at 8 Gy) and JMSD‑medicated serum. PPM1A was overexpressed and knocked down in the AECs via lentivirus transduction and the effects of JMSD administration on the key proteins related to TGF‑β1/Smad signaling were measured by western blotting. It was found that radiation decreased the PPM1A expression in the AECs and JMSD‑medicated serum upregulated the PPM1A expressions in the radiation‑induced AECs. PPM1A overexpression increased the E‑cadherin level but decreased the phosphorylated (p‑)Smad2/3, vimentin and α‑smooth muscle actin (α‑SMA) levels in the AECs. By contrast, the PPM1A knockdown decreased the E‑cadherin level and increased the p‑Smad2/3, vimentin and α‑SMA levels in the AECs and these effects could be blocked by SB431542 (TGF‑β1/Smad signaling inhibitor). JMSD administration increased the E‑cadherin level and decreased the p‑Smad2/3, vimentin and α‑SMA levels in the AECs; however, these effects could be blocked by siPPM1A‑2. In conclusion, PPM1A is a key target of JMSD administration for the attenuation of the radiation‑induced EMT in primary type II AECs via the TGF‑β1/Smad pathway.

More and more evidences show that TGF- β has a crucial role in tumor initiation and development. However, the mechanism of the TGF- β signal regulator in esophageal cancer (EC) is still unclear. Here, we use a variety of bioinformatics methods to analyze the expression and survival data of TGF- β signal regulators in patients with EC. We extracted the expression of the S-TGF- β signal regulator from The Cancer Genome Atlas (TCGA). The cBioPortal database was used to assess the frequency of genetic variation. The TGF- β signal regulator is expressed in EC and normal tissues. The objective is to use the Kaplan-Meier plotter database to investigate the prognostic value of TGF- β signal regulators in cancer patients. The DAVID and clusterProfiler software package were used for functional enrichment analysis. We found that patients with TGF- β signaling mutations have shorter overall survival, disease-free survival, disease-specific survival, platinum overall survival, and platinum-free progression survival. We found that compared with the noncancerous tissues of patients with EC, ZFYVE9, BMPR1B, TGFB3, TGFBRAP1, ACVRL1, TGFBR2, SMAD4, SMAD7, ACVR2A, BMPR1, and SMAD9 were significantly downregulated in tumor tissues, while ACVR1 and Smad1 were significantly upregulated in tumor samples. Univariate survival analysis showed that ACVR1, TGFBR3, TGFBRAP1, BMPR1A, SMAD4, and TGFBR2 were positively correlated with overall survival (OS) prolongation. In addition, TGF- β signal transduction regulators could be divided into two classes. Subclass 1 was involved in regulating cell adhesion, PI3K-Akt signaling, and Rap1 signaling. Subclass 2 was related to regulating angiogenesis and PI3K signaling. In short, all members of TGF- β signal regulators can be used as biomarkers to predict the prognosis of patients with EC., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2021 Wei Song et al.)

[Figure: see text].

Purpose: Urinary bladder urothelial carcinoma (UBUC) is a common malignant disease, and its high recurrence rates impose a heavy clinical burden. The objective of this study was to identify signaling pathways downstream of epithelial membrane protein 2 (EMP2), which induces cytostasis and apoptosis in UBUC., Methods: A series of in vitro and in vivo assays using different UBUC-derived cell lines and mouse xenograft models were performed, respectively. In addition, primary UBUC specimens were evaluated by immunohistochemistry., Results: Exogenous expression of EMP2 in J82 UBUC cells significantly decreased DNA replication and altered the expression levels of several TGFβ signaling-related proteins. EMP2 knockdown in BFTC905 UBUC cells resulted in opposite effects. EMP2-dysregulated cell cycle progression was found to be mediated by the TGFβ/TGFBR1/SP1 family member SMAD. EMP2 or purinergic receptor P2X7 (P2RX7) gene expression upregulation induced apoptosis via both intrinsic and extrinsic pathways. In 242 UBUC patient samples, P2RX7 protein levels were found to be significantly and positively correlated with EMP2 protein levels. Low P2RX7 levels conferred poor disease-specific and metastasis-free survival rates, and significantly decreased apoptotic cell rates. EMP2 was found to physically interact with P2RX7. In the presence of a P2RX7 agonist, BzATP, overexpression of both EMP2 and P2RX7 significantly increased apoptotic cell rates compared to overexpression of EMP2 or P2RX7 alone., Conclusions: EMP2 induces cytostasis via the TGFβ/SMAD/SP1 axis and recruits P2RX7 to enhance apoptosis in UBUC. Our data provide new insights that may be employed for the design of UBUC targeting therapies., (© 2021. Springer Nature Switzerland AG.)

Smads are involved in a variety of biological activities by mediating bone morphogenetic protein (BMP) signals. The full-length coding sequences (CDSs) of buffalo Smads 1 , 4 , and 5 were isolated and identified through RT-PCR in this study. Their lengths are 1398 bp, 1662 bp, and 1398 bp, respectively. In silico analysis showed that their transcriptional region structures, as well as their amino acid sequences, physicochemical characteristics, motifs, conserved domains, and three-dimensional structures of their encoded proteins are highly consistent with their counterparts in the species of Bovidae. The three Smad proteins are all hydrophilic without the signal peptides and transmembrane regions. Each of them has an MH1 domain and an MH2 domain. A nuclear localization sequence was found in the MH1 domain of buffalo Smads 1 and 5 . Prediction showed that the function of the three Smads is mainly protein binding, and they can interact with BMPs and their receptors. The three genes were expressed in all 10 buffalo tissues assayed, and their expression in the mammary gland, gonad, and spleen was relatively high. The results here indicate that the three buffalo Smads may be involved in the transcriptional regulation of genes in a variety of tissues.

The delayed healing response of diabetic wounds is a major challenge for treatment. Negative pressure wound therapy (NPWT) has been widely used to treat chronic wounds. However, it usually requires a long treatment time and results in directional growth of wound healing skin tissue. We investigated whether nonthermal microplasma (MP) treatment can promote the healing of skin wounds in diabetic mice. Splint excision wounds were created on diabetic mice, and various wound healing parameters were compared among MP treatment, NPWT, and control groups. Quantitative analysis of the re-epithelialization percentage by detecting Ki67 and DSG1 expression in the extending epidermal tongue (EET) of the wound area and the epidermal proliferation index (EPI) was subsequently performed. Both treatments promoted wound healing by enhancing wound closure kinetics and wound bed blood flow; this was confirmed through histological analysis and optical coherence tomography. Both treatments also increased Ki67 and DSG1 expression in the EET of the wound area and the EPI to enhance re-epithelialization. Increased Smad2/3/4 mRNA expression was observed in the epidermis layer of wounds, particularly after MP treatment. The results suggest that the Smad-dependent transforming growth factor β signaling contributes to the enhancement of re-epithelialization after MP treatment with an appropriate exposure time. Overall, a short-term MP treatment (applied for 30 s twice a day) demonstrated comparable or better efficacy to conventional NPWT (applied for 4 h once a day) in promoting wound healing in diabetic mice. Thus, MP treatment exhibits promise for treating diabetic wounds clinically.

Autophagy is a key metabolic process where cells can recycle its proteins and organelles to regenerate its own cellular building blocks. Chemotherapy is indispensable for cancer treatment but associated with various side-effects, including organ damage. Stem cell-based therapy is a promising approach for reducing chemotherapeutic side effects, however, one of its main culprits is the poor survival of transplanted stem cells in damaged tissues. Here, we aimed to test the effects of activating autophagy in adipose-derived mesenchymal stem/stromal cells (ADSCs) on the survival of ADSCs, and their therapeutic value in cisplatin-induced liver injury model. Autophagy was activated in ADSCs by rapamycin (50 nM/L) for two hours before transplantation and were compared to non-preconditioned ADSCs. Rapamycin preconditioning resulted in activated autophagy and improved survival of ADSCs achieved by increased autophagosomes, upregulated autophagy-specific LC3-II gene, decreased protein degradation/ubiquitination by downregulated p62 gene, downregulated mTOR gene, and finally, upregulated antiapoptotic BCL-2 gene. In addition, autophagic ADSCs transplantation in the cisplatin liver injury model, liver biochemical parameters (AST, ALT and albumin), lipid peroxidation (MDA), antioxidant profile (SOD and GPX) and histopathological picture were improved, approaching near-normal conditions. These promising autophagic ADSCs effects were achieved by modulation of components in TGF-β1/Smad and PI3K-AKT signaling pathways, besides reducing NF-κB gene expression (marker for inflammation), reducing TGF-β1 levels (marker for fibrosis) and increasing SDF-1 levels (liver regeneration marker) in liver. Therefore, current results highlight the importance of autophagy in augmenting the therapeutic potential of stem cell therapy in alleviating cisplatin-associated liver damage and opens the path for improved cell-based therapies, in general, and with chemotherapeutics, in particular.

Background: Transforming growth factor-β (TGF-β)-induced Epithelial-to-mesenchymal transition (EMT) process is a fundamental target for preventing cervical cancer cells' progression and invasion. Green tea and its principal active substance, Epigallocatechin-3-gallate (EGCG), demonstrate anti-tumor activities in various tumor cells., Methods: The cell viability of two cervical cancer cell lines, Hela and SiHa, in the experimental groups was examined employing the MTT method, and ROS generation was probed applying 2',7'-dichlorofluorescein diacetate-based assay. The Smad signaling and EMT process was evaluated utilizing western blot analysis and quantitative real-time polymerase chain reaction (qRT-PCR). Chromatin immunoprecipitation (ChIP) and Smad binding element (SBE)-luciferase assays were employed to measure Smad-DNA interaction and Smad transcriptional activity, respectively., Results: EGCG (0-100 μmol/L) and green tea extract (0-250 μg/ml) suppressed the viability of cancer cells in a dose-dependent manner (p < 0.01). Our conclusions affirmed that pre-incubation with green tea extract (80 μg/ml) and EGCG (60 μmol/L) significantly reversed the impacts of TGF-β in Hela and SiHa cells by decreasing Vimentin, ZEB, Slug, Snail, and Twist and increasing E-cadherin expression. The molecular mechanism of green tea extract and EGCG for TGF-β-induced EMT inhibition interfered with ROS generation and Smad signaling. Green tea extract and EGCG could significantly decrease ROS levels, the phosphorylation of Smad2/3, the translocation, DNA binding, and activity of Smads in cervical cancer cell lines treated with TGF-β1 (p < 0.01)., Conclusion: EGCG and green tea extract suppressed TGF-β-induced EMT in Hela and SiHa cells, and the underlying molecular mechanism may be related to the ROS generation and Smad signaling pathway., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Transcription factor small mothers against decapentaplegic (Smad) family SMAD proteins are the essential intracellular signal mediators and transcription factors for transforming growth factor β (TGF-β) signal transduction pathway, which usually exert pleiotropic actions on cell physiology, including immune response, cell migration and differentiation. In this study, the Smad family was identified in the most primitive vertebrates through the investigation of the transcriptome data of lampreys. The topology of phylogenetic tree showed that the four Smads (Smad1, Smad3, Smad4 and Smad6) in lampreys were subdivided into four different groups. Meanwhile, homology analysis indicated that most Smads were conserved with typical Mad Homology (MH) 1 and MH2 domains. In addition, Lethenteron reissneri Smads (Lr-Smads) adopted general Smads folding structure and had high tertiary structural similarity with human Smads (H-Smads). Genomic synteny analysis revealed that the large-scale duplication blocks were not found in lamprey genome and neighbor genes of lamprey Smads presented dramatic differences compared with jawed vertebrates. Importantly, quantitative real-time PCR analysis demonstrated that Smads were widely expressed in lamprey, and the expression level of Lr-Smads mRNA was up-regulated with different pathogenic stimulations. Moreover, depending on the weighted gene co-expression network analysis (WGCNA), four Lr-Smads were identified as two meaningful modules (green and gray). The functional analysis of these two modules showed that they might have a correlation with ployI:C. And these genes presented strong positive correlation during the immune response from the results of Pearson's correlation analysis. In conclusion, our results would not only enrich the information of Smad family in jawless vertebrates, but also lay the foundation for immunity in further study., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Genetic alterations affecting transforming growth factor-β (TGF-β) signaling are exceptionally common in diseases and cancers of the gastrointestinal system. As a regulator of tissue renewal, TGF-β signaling and the downstream SMAD-dependent transcriptional events play complex roles in the transition from a noncancerous disease state to cancer in the gastrointestinal tract, liver, and pancreas. Furthermore, this pathway also regulates the stromal cells and the immune system, which may contribute to evasion of the tumors from immune-mediated elimination. Here, we review the involvement of the TGF-β pathway mediated by the transcriptional regulators SMADs in disease progression to cancer in the digestive system. The review integrates human genomic studies with animal models that provide clues toward understanding and managing the complexity of the pathway in disease and cancer., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)