Smad3 Levels Research Articles

Expression and Possible Role of Silent Mating Type Information Regulation 2 Homolog 1 in Post-necrotizing Enterocolitis Stricture in vivo and in vitro.

PurposeTo investigate the expression and possible role of Sirtuin1 or Silent mating–type information regulation 2 homolog-1 (SIRT1) in post-necrotizing enterocolitis stricture.Materials and MethodsThe expression characteristics of SIRT1 and TGF-β1 in post-necrotizing enterocolitis stricture were detected by immunohistochemistry. The siRNA-SIRT1 was used to inhibit the expression of SIRT1 in intestinal epithelial cells-6 (IEC-6), and qRT-PCR, WB, and ELISA were utilized to detect the changes of Transforming growth factor-β1 (TGF-β1), nuclear factor (NF)-κB, tumor necrosis factor-α (TNF-α), tight junction protein-1 (ZO-1), and vascular endothelial growth factor (VEGF) expressions. The IEC-6 cell proliferation and migration ability were tested via CCK8 kit and Transwell test. The expression of E-cadherin and Vimentin in cells was detected by immunofluorescence.ResultsThe CRP, IL-6, IL-10, and IFN-γ in the serum of Necrotizing enterocolitis (NEC) intestinal stenosis patients were significantly higher than the reference values. The SIRT1 protein was under-expressed and the TGF-β1 protein was overexpressed in NEC intestinal stenosis tissue. And the expression of SIRT1 was negatively correlated with TGF-β1. At the time of diagnosis of NEC, the expression of SIRT1 decreased in children with respiratory distress syndrome and CRP level increased. After inhibiting the expression of SIRT1 in IEC6 cells, the expression levels of TGF-β1, Smad3, and NF-κB were decreased, and the expression of ZO-1 was also decreased. The proliferation and migration ability of IEC6 cells was decreased significantly, and the expression of E-cadherin and Vimentin proteins in IEC6 cells did not change significantly.ConclusionPromotion of intestinal fibrosis by inflammation may be the mechanism of post-necrotizing enterocolitis stricture. SIRT1 may be a protective protein of NEC. The probable mechanism is that SIRT1 can regulate intestinal fibrosis and can protect the intestinal mucosal barrier function to participate in the process of post-necrotizing enterocolitis stricture.

HtrA1L364P leads to cognitive dysfunction and vascular destruction through TGF‐β/Smad signaling pathway in CARASIL model mice

AimsCerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is a life‐threatening, inherited, nonhypertensive arteriole disease of the brain. Therapeutic strategy for CARASIL is limited because its pathogenesis is not clear. We previously reported the first family with CARASIL in China, which involves a high‐temperature requirement serine protease gene mutation (HtrA1L364P). Based on this previous study, we constructed a CARASIL mouse model (Mut‐hHtrA1L364P mouse, hereinafter referred to as Mut). This paper aimed to systematically study the behavior, pathology, and molecular biology of Mut mice and explore the pathogenesis and possible therapeutic strategies of CARASIL.MethodsFood maze and water maze experiments were used in the behavioral studies. Pathological studies were carried out by arteriole labeling staining and electron microscopy. The mRNA and protein expression levels of the key factors of TGF‐β/Smad signaling pathway (TGF‐β, Smad2, Smad3, and Smad4) in the brain of the model mice were detected by immunohistochemistry, real‐time quantitative polymerase chain reaction (RT‐PCR), and Western blot assay.ResultsThe food maze and water maze experiment data showed significant differences between the Mut and wild‐type (WT) mice in the first time to find food, the time to contact the escape table for the first time, and the number of times to travel in the escape table quadrant (p < 0.001). The results of vascular labeling staining showed that some small arteries in the brain of Mut mice lost normal structure. The results of electron microscopy showed that the cell morphologies in the cortex and hippocampus of Mut mice were abnormal; the number of synapses was reduced; the walls of capillaries, venules, and arterioles thickened; lumen stenosis and other abnormal phenomenon occurred; and lipofuscin deposition and autophagosomes were found in the hippocampus. Immunohistochemistry, RT‐PCR, and Western Blot results showed that the mRNA and protein expression levels of TGF‐β, Smad2, and Smad3 in the brain of Mut mice increased to different degrees.ConclusionsThe most significant innovation of this study is the first study on the pathogenesis of CARASIL disease using model animals. The Mut mice can well simulate the pathogenesis of CARASIL in behavioral and pathological aspects. The TGF‐β/Smad signaling pathway, which is involved in the pathogenesis of CARASIL, is abnormally upregulated in the brain of Mut mice.

Combination of Sophora flavescens alkaloids and Panax quinquefolium saponins modulates different stages of experimental autoimmune myocarditis via the NF-κB and TGF-β1 pathways.

Chronic cardiac inflammation and fibrosis can progress into severe forms of cardiomyopathy. Sophora flavescens alkaloids (KuShen) have been previously reported to exert anti-inflammatory effects, whereas Panax quinquefolium saponins (XiYangShen) has been shown to alleviate cardiac fibrosis. Therefore, the potential effects of their combination (KX) on different stages of autoimmune myocarditis were investigated in the present study. Mice were randomly divided into the following four groups: Control; experimental autoimmune myocarditis (EAM); KX-High (275 mg/kg); and KX-Low (138 mg/kg). A 21-day and a 60-day EAM model was established through multi-site subcutaneous injections of cardiac myosin mixed with complete Freund's adjuvant on days 0, 7, 21 and 42. Mice in the High and Low KX groups were treated by gavage (10 ml/kg) daily from day 0 (1 day before treatment) until sacrifice (day 21 or 60). Mice in the control and EAM groups received an equivalent volume of distilled water. The levels of lactate dehydrogenase (LDH), creatine kinase-myocardial band (CK-MB), cardiac troponin I (cTn-I), IL-1β, IL-6, TNF-α, TGF-β1, collagen type I (Col Ⅰ) and collagen type III (Col Ⅲ) were measured by ELISA in the mouse myocardial tissues or serum. Myocardial tissue structure and extent of fibrosis were visualized using H&E and Masson's staining. Western blotting and immunohistochemistry were used to measure the expression levels NF-κB and TGF-β1 pathway proteins in the myocardial tissues. The degree of inflammation in the 21-day EAM model was found to be significantly higher compared with that in the 60-day EAM model. KX significantly reduced the inflammatory response at 21 days by decreasing the expression levels of CK-MB, LDH, cTn-I, IL-1β, IL-6, TNF-α and TGF-β-activated kinase 1-binding protein 1/NF-κB pathway proteins. Myocardial fibrosis in the 60-day EAM model was also significantly worse compared with that in the 21-day EAM model. However, fibrosis was significantly delayed by treatment with KX. In addition, KX significantly decreased the expression levels of TGF-β1, Smad2, Smad4, Col I and Col III. Therefore, these data suggest that KX is beneficial for treating myocarditis by targeting multiple pathways.

MiR-208b/miR-21 Promotes the Progression of Cardiac Fibrosis Through the Activation of the TGF-β1/Smad-3 Signaling Pathway: An in vitro and in vivo Study.

BackgroundRegulatory molecule microRNAs (miRNAs) have been implicated in myocardial fibrosis. However, the specific mechanism by which they lead to myocardial fibrosis remains unclear. This study aimed to explore the roles of miR-208b, miR-21 and transforming growth factor-β1 (TGF-β1)/Smad-3 signaling pathway components in cardiac fibrosis development.Materials and MethodsThirty-six consecutive acute myocardial infarction (AMI) patients were included in this study. Plasma was collected on admission and at 24 h, 48 h and 6 d. The levels of plasma miR-208b, miR-21, TGF-β1, and Smad-3 were measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and cardiac calcium protein T (cTnT) and creatine kinase isoenzyme (CK-MB) were detected by electrochemiluminescence analysis. H9C2 cells were exposed to hypoxia and divided into 4 groups (hypoxia treatment for 6 h, 24 h, 48 h, and 72 h). These stimulated cells were then transfected with miRNA inhibitors and mimics for gene overexpression and inhibition. RT-qPCR was used to detect the expression of miR-208b, miR-21, TGF-β1, and Smad-3, and western blot analysis was used to detect TGF-β1 and Smad-3 protein expression.ResultsThe plasma analysis showed cTnT and CK-MB expression peaked at 24 h after symptom onset; miR-208b, miR-21, TGF-β1, and Smad-3 levels showed no peak and increased gradually with time. Cell experiments revealed that miR-208b and TGF-β1 were upregulated along with increased hypoxia exposure; miR-21 expression peaked at 24 h and 72 h, with the highest peak at 72 h, and Smad-3 expression peaked at 6 h and 72 h, with the highest peak at 72 h. miR-208b and miR-21 expressions were positively correlated with TGF-β/Smad-3 expression. TGF-β1/Smad-3 mRNA and protein levels were elevated in the miR-208b and miR-21 overexpression groups and reduced in the miR-208b and miR-21 inhibition groups.ConclusionMiR-208b and miR-21 promote cardiac fibrosis progression through TGF-β1/Smad-3 signaling pathway activation.

Cinobufacini Inhibits the Development of Pancreatic Cancer Cells through the TGFβ/Smads Pathway of Pancreatic Stellate Cells.

This study aimed to test cinobufacini therapeutic potential for pancreatic cancer, verify its potential molecular mechanism, and evaluate the cinobufacini impact on pancreatic cancer microenvironment. First, the effect of cinobufacini-treated pancreatic stellate cells (PSCs) supernatant on the value-added ability of pancreatic cancer (PCCs) was tested. The results show that cinobufacini can effectively reduce the ability of PSCs supernatant to promote the value-added PCCs. Further results show that cinobufacini can effectively reduce the concentration of TGFβ in the supernatant of PSCs. Subsequently, the impact of cinobufacini on the transcription and translation levels of key genes in the TGFβ/Smads pathway was examined. The results showed that the impact of cinobufacini on the transcription levels of Smad2, Smad3, and Smad7 was in a concentration-dependent manner, while the transcriptional activity of collagen I mRNA was decreased with the increase of cinobufacini concentration. The results of protein expression showed that cinobufacini could upregulate the expression of inhibitory protein Smad7, inhibit the phosphorylation level of p-Smad2/3, and then suppress the expression of type I collagen (collagen I). On the one hand, this study shows that cinobufacini can inhibit the promotion of PSCs on the proliferation of PCCs. On the other hand, cinobufacini can upregulate the expression of the inhibitory molecule, Smad7, through the TGFβ/Smads pathway and reduce the phosphorylation level of p-Smad2/3, thereby inhibiting the expression of collagen I and pancreatic fibrosis. cinobufacin can inhibit the proliferation of SW1900 cells by blocking the TGFβ/Smads pathway of pancreatic stellate cells. These results provide a clinical basis for the treatment of pancreatic cancer.

Pseudogene MSTO2P Interacts with miR-128-3p to Regulate Coptisine Sensitivity of Non-Small-Cell Lung Cancer (NSCLC) through TGF-β Signaling and VEGFC.

Background Coptisine has been widely used for treating a variety of cancer types. To date, whether pseudogene is implicated in coptisine resistance of NSCLC remains unknown. Methods We performed MTT to assess the cell viability of A549 and Calu-1 cells. The transwell assay was used to examine the invasion of cells. TUNEL was used to determine apoptosis. Results Our data showed that coptisine treatment suppressed cell viability and invasion of NSCLC cells while contributing to apoptosis. MiR-128-3p negatively regulated MSTO2P. miR-128-3p reverted MSTO2P knockdown-attenuated cell viability and invasion, as well as promoted cell apoptosis of A549 cells. Moreover, we identified TGF-β signaling and VEGFC as key downstream effectors for MSTO2P and miR-128-3p in A549 cells. MiR-128-3p mimic inhibited TGF-β pathway-associated genes (TGFBR1, Smad2, Smad5, and Smad9), whereas miR-128-3p inhibitor exerted opposite effect. MSTO2P knockdown led to attenuated expression levels of TGFBR1, Smad2, Smad5 and Smad9. VEGFC overexpression greatly rescued miR-128-3p-modulated cell viability, invasion, and apoptosis of A549 cells. Conclusion MSTO2P plays a role in coptisine therapy of NSCLC through miR-128-3p. The findings will advance our understanding of NSCLC treatment.

Expression and role of miR-146a and SMAD4 in placental tissue of pregnant women with preeclampsia.

To investigate the expression of miR-146a in severe preeclampsia (PE) and its effect on trophoblast cell proliferation, invasion and apoptosis, as well as its relationship with SMAD4. Participants were divided into the severe PE group (n=30) and the normal group (n=30). The expression of miR-146a and SMAD4 in placenta tissue was detected by immunohistochemistry, qRT-PCR, and western blot. Trophoblast cell lines HTR-8/SVneo were cultured to detect the expression of miR-146a under the Cobalt chloride (CoCl2 )-simulated hypoxia. The effects of miR-146a transfection on cell proliferation, invasion, apoptosis, and SMAD4 expression were analyzed. Compared with the normal group, miR-146a expression was decreased and the protein and mRNA levels of SMAD4 were increased in placenta tissues of the severe PE group. Our in vitro experiments showed that the expression of miR-146a decreased after CoCl2 treatment. Silencing miR-146a caused increased expression of SMAD4 and decreased expression of VEGF. After transfection with miR-146a inhibitor, compared with the NC group, the invasion and proliferation of HTR-8/Svneo cells were decreased, while the apoptosis was enhanced. The expression of miR-146a decreased in severe PE and was negatively correlated with SMAD4 expression. The expression of miR-146a was inhibited under hypoxia, and the low expression of miR-146a affected the proliferation, invasion, and apoptosis of trophoblast cells.

Abstract 996: Fibronectin exposes a targetable DHPS/SLC3A2 vulnerability that can be leveraged to decrease cytoplasmic levels of eIF5A1/2 and proliferation/survival in TNBC

Abstract Breast cancer ranks first among female cancer-related deaths in 2021, and fewer than 20% of patients survive five years after metastasis. Triple negative breast cancer (TNBC or basal) is highly aggressive and particularly difficult to treat due to the lack therapeutic target receptors. Notably, elevated fibronectin expression within the microenvironment of TNBC is an indicator of advanced disease stage. In this regard, we previously reported that the two-step, hypusination reaction that activates eukaryotic initiation factor 5A 1/2 (eIF5A1/2) is necessary for PEAK1 pseudokinase translation and fibronectin/TGFβ-mediated TNBC metastasis. In our current study, we observed intratumoral heterogeneity of DOHH, DHPS, eIF5A and eIF5A2 expression in TNBC patient tumor samples, suggesting a role for this pathway in disease progression and therapy resistance. Using bioinformatics tools, we identified a correlation between elevated solute carrier 3A2 (SLC3A2 or CD98) and decreased survival in breast cancer patients with amplified SMAD3 loci. Given that SMAD3-dependent transcription is one mechanism by which canonical TGFβ signaling elicits tumor suppressive outcomes, we asked whether SLC3A2 may function to prime TNBC cells for tumor promoting outcomes in response to TGFβ. In this regard, inhibition of SLC3A2 or DHPS decreased TNBC spheroid growth. Notably, spiking fibronectin into 3D culture media partially rescued spheroid growth from SLC3A2 inhibition - an effect blocked by dual DHPS/SLC3A2 inhibition. In agreement with these data, dual DHPS/SLC3A2 inhibition in TNBC cells cultured with exogenous fibronectin significantly reduced the percentage of cells in S phase and subsequently increased the percentage of cells in either G0/G1 or Sub-G0/G1 phases, suggesting that DHPS and SLC3A2 cooperatively promote TNBC cell proliferation/survival in fibronectin-rich contexts. eIF5A1/2 carries out hypusination-dependent functions in both the nucleus (i.e., mRNA export) and cytoplasm (i.e., translation). Interestingly, it undergoes nucleocytoplasmic transport via exportin four (XPO4) - the same nuclear pore complex protein that is used for nuclear export of SMAD3. Using structured illumination microscopy, we discovered that fibronectin promotes an increase in cytoplasmic eIF5A1/2 levels - an effect blocked by dual DHPS/SLC3A2 inhibition. In contrast, DHPS inhibition alone increased nuclear SMAD3 levels independent of extracellular matrix proteins. Taken together, these data provide a possible mechanism for the antiproliferative/cytotoxic effects of dual DHPS/SLC3A2 inhibition and suggest that this treatment strategy may be efficacious in TNBC patients with advanced stage disease. Citation Format: Cameron Geller, Joanna Maddela, Ranel Tuplano, Joseph Cantor, Jonathan A. Kelber. Fibronectin exposes a targetable DHPS/SLC3A2 vulnerability that can be leveraged to decrease cytoplasmic levels of eIF5A1/2 and proliferation/survival in TNBC [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 996.

BMP4 Regulates EMT to be Involved in non-Syndromic Cleft lip With or Without Palate.

In the previous study, we identified bone morphogenetic protein 4 (BMP4) responsible for non-syndromic cleft lip with or without cleft palate (NSCL/P). We aimed to elucidate the effects and mechanisms of BMP4 on epithelial-mesenchymal transition (EMT) through Smad1 signaling pathway to be involved in NSCL/P. The human oral epidermoid carcinoma cells (KBs) were transfected with plasmids or small interfering RNA (siRNA) to build the models. The migration of the cells was evaluated by transwell assay. Western blotting and quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) were used to detect the expressions of BMP4, E-cadherin, N-cadherin, EMT-related transcription factors snal1 and snal2, matrix metalloproteinase 2 (MMP2), MMP9, Smad1, and phosphorylated Smad1. In the overexpression group, the migration number of cells was increased significantly. The protein expression of E-cadherin was decreased significantly, while the protein expression level of the N-cadherin was increased significantly. The protein and mRNA expressions of MMP2, MMP9, snal1, and snal2 were significantly higher. The expression level of Smad1 was not significantly changed, while the phosphorylation of Smad1 was significantly increased. In the BMP4-siRNA group, the migrating number cells was significantly decreased. The protein expression of E-cadherin was increased significantly, while the expression of N-cadherin was significantly decreased. The protein and mRNA expressions of MMP2, MMP9, snal1, and snal2 were significantly lower than that of the control group. The expressions of Smad1 and phosphorylation of Smad1 were not significantly changed. BMP4 enhances cell migration and promotes cell EMT through Smad1 signaling pathway. Abnormal BMP4 mediates migration and EMT through other relevant signaling pathways resulting in NSCL/P. The study provides new insight into the mechanisms of NSCL/P associated with BMP4.n.

MiR-221-5p/Smad3 axis in osteoclastogenesis and its function: Potential therapeutic target for osteoporosis

ObjectiveTo probe the role of miR-221-5p in osteoclastogenesis and the underlying mechanism. MethodsSerum from patients with postmenopausal osteoporosis and healthy controls was collected for determination of miR-221-5p expression. For in vitro experiment, RAW264.7 macrophages, in which the expression of miR-221-5p and/or Smad3 was altered, were induced by RANKL to differentiate into osteoclasts. For in vivo experiment, ovariectomy was performed to construct osteoporosis mouse models, followed by tail vein injection of miR-221-5p agomir. qRT-PCR and/or western blot were applied to measure the expression of miR-221-5p, Smad3, and osteoclastogenesis-related genes (NFATc1 and TRAF6). TRAP staining was utilized for assessment of osteoclast formation, MTT assay for assessment of osteoclast viability, and H&E staining for observation of histomorphological changes. The targeting relationship between miR-221-5p and Smad3 was verified by dual-luciferase reporter gene assay. ResultsCompared with healthy controls, patients with postmenopausal osteoporosis had decreased miR-221-5p expression and lower lumbar vertebra bone mineral density. MiR-221-5p expression was decreased and Smad3 level was increased during osteoclastogenesis. The osteoclastogenesis was suppressed by miR-221-5p and promoted by Smad3, as evidenced by diminished number and viability of osteoclasts following overexpression of miR-221-5p or knockdown of Smad3. MiR-221-5p negatively mediated Smad3 expression. Smad3 suppression nullified the pro-osteoclastogenesis effect of miR-221-5p inhibition. Consistent results were observed in osteoporosis mouse models. ConclusionMiR-221-5p may alleviate postmenopausal osteoporosis through suppressing osteoclastogenesis via Smad3, which provides new ideas for molecule-targeted therapy of osteoporosis.

Tanshinone IIA regulates the TGF-β1/Smad signaling pathway to ameliorate non-alcoholic steatohepatitis-related fibrosis.

Tanshinone IIA (TIIA) is a major component extracted from the traditional herbal medicine Salvia miltiorrhiza and has been indicated to play a role in the treatment of organ fibrosis. However, the evidence supporting its antifibrotic effect is insufficient and the underlying mechanism is unclear. To investigate the therapeutic effect of TIIA on non-alcoholic steatohepatitis-related fibrosis (NASH-F), the present study used a methionine choline deficiency diet to induce NASH-F in rats, and explored the effect of TIIA on the transforming growth factor-β1 (TGF-β1)/Smad signaling pathway. Wistar rats were randomly divided into control, NASH-F and TIIA groups. After 8 weeks of treatment, the levels of serum markers associated with liver function and fibrosis were measured, liver fat vacuoles and inflammation were assessed by haematoxylin and eosin staining, and liver fibrosis was assessed by Masson's trichrome staining. TGF-β1, Smad2, Smad3, Smad7 and α-smooth muscle actin (α-SMA) mRNA expression, and TGF-β1, Smad2/3, phosphorylated (p)-Smad2/3, Smad7 and α-SMA protein levels were determined. The results revealed that TIIA could remarkably ameliorate liver fat vacuoles and inflammation in NASH-F rats, and could decrease the levels of serum aspartate aminotransferase, alanine aminotransferase, total bilirubin, total bile acid, hyaluronic acid, type Ⅳ collagen, laminin and type III collagen, while increasing the levels of total cholesterol and triglycerides; however, this was not statistically significance. TIIA markedly suppressed the increased TGF-β1, Smad2, Smad3 and α-SMA mRNA expression levels observed in the liver of NASH-F rats, while it increased the mRNA expression level of Smad7. Similarly, TIIA suppressed the increased TGF-β1, p-Smad2/3 and α-SMA protein levels observed in the liver of NASH-F rats, while it increased the protein expression level of Smad7 in vitro and in vivo. TIIA had no significant cytotoxic effect at 10, 20, 40 and 80 µmol/l on human LX-2 cell. In conclusion, the findings of the present study indicated that TIIA alleviated NASH-F by regulating the TGF-β1/Smad signaling pathway. TIIA may be a useful tool in the prevention and treatment of NASH-F.

β-Elemene Attenuates Renal Fibrosis in the Unilateral Ureteral Obstruction Model by Inhibition of STAT3 and Smad3 Signaling via Suppressing MyD88 Expression.

Renal fibrosis is a chronic pathological process that seriously endangers human health. However, the current therapeutic options for this disease are extremely limited. Previous studies have shown that signaling factors such as JAK2/STAT3, Smad3, and Myd88 play a regulatory role in renal fibrosis, and β-elemene is a plant-derived sesquiterpenoid organic compound that has been shown to have anti-inflammatory, anti-cancer, and immunomodulatory effects. In the present study, the anti-fibrotic effect of β-elemene was demonstrated by in vivo and in vitro experiments. It was shown that β-elemene inhibited the synthesis of extracellular matrix-related proteins in unilateral ureteral obstruction mice, and TGF-β stimulated rat interstitial fibroblast cells, including α-smooth muscle actin, vimentin, and connective tissue growth factor, etc. Further experiments showed that β-elemene reduced the expression levels of the above-mentioned fibrosis-related proteins by blocking the phosphorylation of JAK2/STAT3, Smad3, and the expression or up-regulation of MyD88. Notably, knockdown of MyD88 attenuated the phosphorylation levels of STAT3 and Smad3 in TGF-β stimulated NRK49F cell, which may be a novel molecular mechanism by which β-elemene affects renal interstitial fibrosis. In conclusion, this study elucidated the anti-interstitial fibrosis effect of β-elemene, which provides a new direction for future research and development of drugs related to chronic kidney disease.

TGF-β/Akt/Smad signaling regulates ionizing radiation-induced epithelial-mesenchymal transition in acquired radioresistant lung cancer cells

ObjectiveTo define the properties of lung cancer cells that resisted conventionally fractionated radiation exposure. MethodsAcquired radioresistant lung cancer cell line A549 was constructed by X-ray irradiation with a clinical conventional fraction dose of 2 ​Gy daily during 30 fractions. Cell morphology, molecular markers, migration capacity and invasion potential were evaluated by the microscope, Western blot, immunofluorescence, wound healing test and transwell chamber assay, respectively. ResultsRadioresistant A549 ​cells shifted from an epithelial to a mesenchymal morphology, termed as epithelial-mesenchymal transition (EMT), and was accompanied by decreased expressions of epithelial markers (F ​= ​4.568, P ​< ​0.05) and increased expression of mesenchymal markers (F ​= ​4.270, P ​< ​0.05), greater migratory and invasive capabilities (t ​= ​6.386, 5.644, P ​< ​0.05). The expression of TGF-β, and phosphorylated levels of Akt and Smad3 were also enhanced (F ​= ​6.496, 4.685, 3.370, P ​< ​0.05). Furthermore, the EMT phenotype induced by radiation could be reversed through inhibition of TGF-β, Akt or Smad3, indicating a functional relationship between them. ConclusionsEMT mediates acquired radioresistance of lung cancer cells induced by IR with clinical parameters, and the crosstalk mode of TGF-β/Akt/Smad signaling plays a critical regulatory role in this process.

Piezo1 Affects Temporomandibular Joint Osteoarthritis by Influencing pSmad3.

Objective: The aim of this research was to study the expression of Piezo1 in a rat temporomandibular joint osteoarthritis animal model and to explore its mechanism for inducing inflammatory changes. Methods: A total of 24male SD rats aged approximately 8 weeks were randomly divided into three groups: the blank control group, complete Freund’s adjuvant group (CFA), and CFA + inhibitor (GsMTx4) group. After 3 weeks, the condylar heads of the rats were evaluated by micro-CT, HE, immunohistochemistry, safranin O staining, and other experimental techniques. Protein was extracted from the subchondral bone, and the changes in Piezo1, Smad3, and pSmad3 levels in each group were detected by Western blotting. p < 0.05 was considered to indicate statistical significance. Results: The degree of damage to the cartilage and subchondral bone in the Piezo1 inhibitor group was smaller than that in the CFA group. The expression level of Piezo1 in the CFA group was higher than that in the other groups, and the difference was statistically significant. The expression of pSmad3 in the CFA group was also higher than that in the other groups (p < 0.05). Conclusion: Piezo1 is expressed in the condylar cartilage and subchondral bone of rats, and the degree of condylar destruction can be improved by influencing the pSmad3 expression.

MO425: Rosuvastatin Activates Hox13-Usag-1 Pathway and Prevents Renal Fibrosis

Abstract BACKGROUND AND AIMS Statins, 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, are primarily cholesterol-lowering drugs that have become standard of care in the primary and secondary prevention of cardiovascular diseases. Apart from lipid-lowering, statins may act beneficially through anti-fibrotic mechanisms to protect the diseased kidney. This study evaluated anti-fibrotic effects of rosuvastatin (RSV) in a chronic kidney fibrosis model and against the TGF-ß1 stimulated Madin-Darby canine kidney (MDCK) cells in vitro. METHOD Mice subjected to unilateral ischemic reperfusion injury with contralateral nephrectomy (uIRIx) were treated with vehicle or RSV (10 mg/kg, by oral gavage) daily for 4 weeks and kidneys were analyzed for markers of fibrosis, bone morphogenetic protein-7 (BMP-7), uterine sensitization-associated gene-1 (USAG-1), and SMAD signaling. Control and homeobox protein Hox-A13 (HOXA13) knocked down MDCK cells were stimulated with TGF- ß1 (5 ng/ml) and then treated with RSV. RESULTS Kidneys from uIRIx mice showed increased expression of α-SMA, Collagen 1 and decrease in BMP-7 (20.35 ± 2.37 versus 1.00 ± 0.27, P &amp;lt; 0.05; 8.43 ± 1.55 versus 1.00 ± 0.35, P &amp;lt; 0.05; 0.75 ± 0.06 versus 1.00 ± 0.15, P &amp;lt; 0.05, respectively). In contrast, expression of USAG-1, a BMP-7 antagonist, was markedly increased in fibrotic kidney (6.60 ± 1.11 versus 1.00 ± 0.02, P &amp;lt; 0.05). Interestingly, RSV treatment not only attenuated expression of USAG-1 (0.72 ± 0.08 versus 1.36 ± 0.14, P &amp;lt; 0.05) but also showed a tendency to activate expression of HOXA13 (0.36 ± 0.14 versus 0.51 ± 0.15, P &amp;gt; 0.05) and improved other markers of fibrosis. Moreover, RSV treatment significantly reduced phosphorylated Smad3 (3.94 ± 0.81 versus 7.17 ± 1.50, P &amp;lt; 0.05) and increased phosphorylation levels of Smad 1/5/9 (0.67 ± 0.10 versus 0.30 ± 0.09, P &amp;lt; 0.05) that is associated with BMP-7 signaling in the fibrotic kidney. MDCK cells stimulated with TGF-β1 in vitro showed increased expression of α-SMA, fibronectin, vimentin, collagen 1, USAG-1, and phosphorylation of Smad3 as well as decreased expression of phosphorylated Smad 1/5/9. RSV treatment significantly reversed these changes as well as increased level of transcriptional factor HOXA13 (0.89 ± 0.12 versus 0.33 ± 0.08, P &amp;lt; 0.05), which negatively regulates USAG-1, without changes in BMP-7 expression. In addition, effect of RSV treatment on USAG-1 expression was significantly decreased in HOXA13 gene knocked down MDCK cells (1.01 ± 0.19 versus 2.04 ± 0.38, P &amp;lt; 0.05; 1.66 ± 0.13 versus 2.04 ± 0.38, P &amp;gt; 0.05; TGF- ß1 + RSV versus TGF- ß1 and TGF- ß1 + RSV + siRNA vs TGF- ß1, respectively). These in vitro data suggest that RSV enhanced anti-fibrotic pathway by downregulating a dominant BMP-7 antagonist USAG-1 via HOXA13 upregulation and not by enhancing BMP-7 production. CONCLUSION The present results demonstrate that RSV inhibits the progression of kidney fibrosis in part by upregulating BMP-7-mediated signaling via HOXA13 expression and down regulation of USAG-1.

Epigenetic Modulation of Class-switch DNA Recombination to IgA by miR- 146a through Downregulation of Smad2, Smad3 and Smad4

Abstract IgA is the predominant isotype at intestinal mucosae, where it plays a critical role in homeostasis and first line of immune protection. Class-switch DNA recombination (CSR) to IgA is directed by TGF-b which induces activation of Smad2 and Smad3 transcription factors. Activated Smad2/Smad3 dimers are recruited with Smad4 to IgH α locus Iα promoter to activate germline Iα-Cα transcription, the first step in CSR to IgA. Epigenetic factors, such as microRNAs, regulate immune cells and, as we have shown, modulate the antibody response in a B cell- intrinsic fashion. We found that miR-146a targets Smad2, Smad3 and Smad4 mRNA 3’UTRs, and keeps CSR to IgA in check in resting B cells. Enforced miR-146a expression in B cells aborted CSR to IgA by decreasing Smad levels. Induction of CSR to IgA, as directed by TGF-b, downregulated B cell miR-146a, thereby reversing the silencing of Smad2, Smad3 and Smad4, which, once expressed, led to recruitment of Smad2, Smad3 and Smad4 to Iα promoter for activation of germline Iα-Cα transcription. miR- 146a−/− mice significantly increased circulating levels of total IgA, but not IgM, IgG or IgE, and heightened the specific IgA antibody response to OVA. In miR-146a−/− mice, this was associated with increased IgA+ B cells in intestinal mucosae, increased amounts of fecal free and bacteria-bound IgA, kidney IgA deposition and IgA nephropathy. The B cell-intrinsic role of miR-146a in repression of Smad2, Smad3 and Smad4 and CSR to IgA was confirmed in miR-146a −/− B cells in vitro and in vivo in mixed bone marrow μMT/miR-146a−/− chimeric mice. Thus, by specific inhibition of Smad2, Smad3 and Smad4 expression, miR-146a plays an important and B cell intrinsic role in modulation of CSR to IgA and the IgA antibody response. Supported by NIH grants AI 079705, AI 105813, AAI 167416 and LRA grant 641363 to PC.

JPHYD Inhibits miR-21-5p/Smad7-Mediated Epithelial-Mesenchymal Transition of Hepatocellular Carcinoma Cells.

Background Studies have shown that Jianpi Huayu Decoction (JPHYD) can inhibit the growth of hepatocellular carcinoma cells, but the mechanism of its effect was not clear at present. Methods We assessed the effect of JPHYD using liver cancer cells as in vitro cell model and xenograft tumor as in vivo model. CCK8, EdU, wound-healing, and transwell assays were performed to assess the cell growth, migration, and invasion of hepatocellular carcinoma (HCC) cell lines HepG2 and MHCC97H. Western blot assay was performed to observe the protein level of E-cadherin, Smad7, N-cadherin, Snail, Smad3, Vimentin, and Zeb1. qRT-PCR assay was used to observe the expression of miR-21-5p in clinical liver cancer tissue samples and in HepG2 and MHCC97H cells. Animal tumorigenesis experiments and in vivo imaging experiments were performed to assess the results of in vitro experiments. Results We found that JPHYD could inhibit the proliferation, invasion, and migration of hepatocellular carcinoma cells and JPHYD decreased the level of N-cadherin, Snail, Vimentin, Smad3, and Zeb1 and increased E-cadherin and Smad7 proteins. The expression of miR-21-5p was increased while that protein of Smad7 was decreased in HCC tissues. The vivo experiments also showed that miR-21-5p could promote the migration of HCC cells. JPHYD decreased miR-21-5p expression. The same results have been found in animal studies. Conclusion Our results indicated that JPHYD inhibited epithelial-mesenchymal transition by increasing Smad7 expression and inhibiting miR-21-5p. Therefore, blocking the occurrence and development of EMT may be a new mechanism of JPHYD's anti-liver cancer effect.

PELO facilitates PLK1-induced the ubiquitination and degradation of Smad4 and promotes the progression of prostate cancer.

PLK1 and Smad4 are two important factors in prostate cancer initiation and progression. They have been reported to play the opposite role in Pten-deleted mice, one is an oncogene, the other is a tumor suppressor. Moreover, they could reversely regulate the PI3K/AKT/mTOR pathway and the activation of MYC. However, the connections between PLK1 and Smad4 have never been studied. Here, we showed that PLK1 could interact with Smad4 and promote the ubiquitination and degradation of Smad4 in PCa cells. PLK1 and PELO could bind to different domains of Smad4 and formed a protein complex. PELO facilitated the degradation of Smad4 through cooperating with PLK1, thereby resulting in proliferation and metastasis of prostate cancer cell. Changes in protein levels of Smad4 led to the alteration of biological function that caused by PLK1 in prostate cancer cells. Further studies showed that PELO upregulation was positively associated with high grade PCa and knockdown of PELO expression significantly decreased PCa cell proliferation and metastasis in vitro and vivo. PELO knockdown in PCa cells could enhance the tumor suppressive role of PLK1 inhibitor. In addition, blocking the interaction between PELO and Smad4 by using specific peptide could effectively inhibit PCa cell metastasis ability in vitro and vivo. Overall, these findings identified a novel regulatory relationship among PLK1, Smad4 and PELO, and provided a potential therapeutic strategy for advanced PCa therapy by co-targeting PLK1 and PELO.

Mechanism of cleft palate in C57BL/6N mice induced by 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin

Objective: To explore the molecular mechanism of cleft palate in mice induced by 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). Methods: The pregnant mice were randomly divided into TCDD-treated group (n=42) and control group (n=42). TCDD-treated group was given by gavage a single dose of TCDD (64 μg/kg) at 8: 00 AM on gestation day 10 (GD10) and the control group was given by gavage the isopyknic corn oil. At GD13-GD15, the fetal mice palate development was observed by HE staining. The mouse embryonic palatal mesenchymal cell proliferation was detected by 5-bromo-2-deoxyuridine (BrdU) immunofluorescence. The localization and expression of maternally expressed gene3 (MEG3) in mouse embryonic palatal mesenchymal cells was detected by situ hybridization and real-time PCR (RT-PCR). The key protein expressions of transforming growth factor-β (TGF-β)/Smad signaling pathway in mouse embryonic palatal mesenchyme were analyzed by Western blotting. The interaction of MEG3 and TGF-β receptor Ⅰ (TGF-βRⅠ) was examined by RNA binding protein immunoprecipitation (RIP). Results: At GD13 and GD14, compared with the control group, the ratio of BrdU-positive cells in the palatal mesenchyme of TCDD-treated fetuses decreased significantly (GD13, t=6.66, P=0.003; GD14, t=6.56, P=0.003). However, at GD15, the ratio of BrdU-positive cells was significantly increased (t=-5.98, P=0.004). MEG3 was mainly expressed in the nuclei of fetal mouse palatal mesenchymal cells, and the expression of MEG3 in TCDD group was significantly increased at GD13, GD14 and GD15(GD13, t=39.28, P=0.012; GD14, t=18.75, P=0.042; GD15, t=28.36, P=0.045). At GD14, TCDD decreased the levels of p-Smad2 and Smad4 in embryonic palate mesenchymal cells (p-Smad2, t=9.48, P=0.001;Smad4, t=63.10, P=0.001), whereas the expression of Smad7 was significantly increased at GD14 (t=30.77, P<0.001). The results of the RIP experiment showed that the amount of TGF-βRⅠ-bound MEG3 in mouse embryonic palatal mesenchymal cells in the TCDD group (23.940±1.301) was higher than that in the control group (8.537±1.523)(t=24.55, P<0.001). Conclusions: MEG3 is involved in the suppression of mouse embryonic palatal mesenchymal cell proliferation, functioning at least in part via interacting with the TGF-βRⅠ protein and thereby suppressing Smad signaling in the context of TCDD induced cleft palate.

Guizhi Fuling pill attenuates liver fibrosis in vitro and in vivo via inhibiting TGF-β1/Smad2/3 and activating IFN-γ/Smad7 signaling pathways

ABSTRACT Liver fibrosis resulting from chronic liver injuries (CLI) is a common health problem globally. Guizhi Fuling pill (GZFL), a modern preparation from traditional Chinese medicine, exhibited anti-dysmenorrhea, anti-inflammatory, and immune-regulative effects. However, the effect of GZFL on liver fibrosis remains unknown. In this research, LX-2 cells were stimulated with acetaldehyde for mimicking liver fibrosis progression in vitro. In addition, carbon tetrachloride (CCl4)-induced mouse model of liver fibrosis was established as well. The data revealed GZFL obviously suppressed the proliferation and triggered the apoptosis of acetaldehyde-stimulated LX-2 cells. In addition, GZFL prevented acetaldehyde-induced activation of LX-2 cells via downregulation of TGF-β1, p-Smad2, p-Smad3, CUGBP1, and upregulation of p-STAT1 and Smad7. Meanwhile, GZFL significantly alleviated CCl4‑induced liver fibrosis, as evidenced by the decrease of ALT and AST levels. Moreover, GZFL downregulated the expressions of TGF-β1, p-Smad2, p-Smad3, and CUGBP1 in CCl4-treated mice. Furthermore, GZFL remarkably elevated the levels of IFN-γ, p-STAT1, and Smad7 in CCl4-treated mice. To sum up, GZFL was able to inhibit liver fibrosis in vitro and in vivo through suppressing TGF-β1/Smad2/3-CUGBP1 signaling and activating IFN-γ/STAT1/Smad7 signaling. Thus, GZFL might have a potential to act as a therapeutic agent for anti-fibrotic therapy.